U.S. patent application number 10/602853 was filed with the patent office on 2004-01-15 for vectors for tissue-specific replication and gene expression.
This patent application is currently assigned to Genetic Therapy, Inc.. Invention is credited to Chang, Yung-Nien, Hallenbeck, Paul L., Hay, Carl M., Stewart, David A..
Application Number | 20040009588 10/602853 |
Document ID | / |
Family ID | 25521982 |
Filed Date | 2004-01-15 |
United States Patent
Application |
20040009588 |
Kind Code |
A1 |
Chang, Yung-Nien ; et
al. |
January 15, 2004 |
Vectors for tissue-specific replication and gene expression
Abstract
The invention generally relates to cell-specific expression
vectors. It particularly relates to targeted gene therapy using
recombinant expression vectors and particularly adenovirus vectors.
The invention specifically relates to replication-conditional
expression vectors and methods for using them. Such vectors are
able to selectively replicate in a target cell or tissue to provide
a therapeutic benefit in a tissue from the presence of the vector
per se or from one or more heterologous gene products expressed
from the vector and distributed throughout the tissue. In such
vectors, a gene essential for replication is placed under the
control of a heterologous tissue-specific transcriptional
regulatory sequence. Thus, replication is conditioned on the
presence of a factor(s) that induces transcription or the absence
of a factor(s) that inhibits transcription of the gene by means of
the transcriptional regulatory sequence with this vector;
therefore, a target tissue can be selectively treated.
Inventors: |
Chang, Yung-Nien;
(Cockysville, MD) ; Hallenbeck, Paul L.;
(Gaithersburg, MD) ; Hay, Carl M.; (Damascus,
MD) ; Stewart, David A.; (Eldersburg, MD) |
Correspondence
Address: |
THOMAS HOXIE
NOVARTIS, CORPORATE INTELLECTUAL PROPERTY
ONE HEALTH PLAZA 430/2
EAST HANOVER
NJ
07936-1080
US
|
Assignee: |
Genetic Therapy, Inc.
|
Family ID: |
25521982 |
Appl. No.: |
10/602853 |
Filed: |
June 24, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10602853 |
Jun 24, 2003 |
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08974391 |
Nov 19, 1997 |
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6638762 |
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08974391 |
Nov 19, 1997 |
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08487992 |
Jun 7, 1995 |
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08487992 |
Jun 7, 1995 |
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08348258 |
Nov 28, 1994 |
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08849117 |
Aug 1, 1997 |
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PCT/US95/15455 |
Nov 28, 1995 |
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PCT/US95/15455 |
Nov 28, 1995 |
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08487992 |
Jun 7, 1995 |
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08487992 |
Jun 7, 1995 |
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08348258 |
Nov 28, 1994 |
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Current U.S.
Class: |
435/320.1 ;
514/44R |
Current CPC
Class: |
C12N 2830/30 20130101;
C12N 2840/44 20130101; C12N 2840/20 20130101; C12N 2830/008
20130101; A61P 35/00 20180101; C12N 15/86 20130101; A61K 48/00
20130101; C12N 2710/10343 20130101; C12N 2830/85 20130101; C12N
2830/15 20130101; C12N 2830/32 20130101 |
Class at
Publication: |
435/320.1 ;
514/44 |
International
Class: |
A61K 048/00; C12N
015/00 |
Claims
What is claimed is:
1. An expression vector comprising: (a) a first coding sequence
operably linked to a tissue-specific transcriptional regulatory
sequence wherein the gene product of said first coding sequence is
essential for vector replication, wherein said first coding
sequence and said tissue-specific transcriptional regulatory
sequence are not derived from the same gene; and (b) at least one
additional coding sequence encoding a heterologous gene product,
wherein said additional coding sequence is operably linked to said
tissue-specific transcriptional regulatory sequence or is operably
linked to a second transcriptional regulatory sequence that is
activated by the gene product of said first coding sequence.
2. The vector of claim 1, wherein said first and at least one of
said additional coding sequences are transcribed from a single
tissue-specific transcriptional regulatory sequence.
3. The vector of claim 1, wherein said first coding sequence and at
least one of said additional coding sequences are transcribed from
separate tissue-specific transcriptional regulatory sequences.
4. The vector of claim 1, wherein at least one of said additional
coding sequences replaces a coding sequence in a gene on the
vector, which gene is not essential for vector replication, so that
said additional coding sequence is operably linked to and
transcribed from the transcriptional regulatory sequence from the
gene nonessential for vector replication.
5. The vector of claim 1, wherein said tissue-specific
transcriptional regulatory sequence is a promoter or an
enhancer.
6. The vector of claim 5, where said promoter is selected from the
group consisting of CEA, MUC1/DF3, .alpha.-fetoprotein, erb-B2,
surfactant, tyrosinase, PSA, TK, p21, and cyclin.
7. The vector of claim 5, wherein said enhancer is selected from
the group consisting of DF3, breast cancer-specific enhancer, viral
enhancers, and steroid receptor enhancers.
8. The vector of claim 1, wherein said additional coding sequence
is selected from the group consisting of thymidine kinase, cytosine
deaminase, and purine nucleoside phosphorylase.
9. The vector of claim 1, wherein said vector is a DNA virus.
10. The vector of claim 9, wherein said DNA virus is selected from
the group consisting of adenovirus, herpesvirus, papovavirus,
papillomavirus, and hepatitis virus.
11. The vector of claim 10, wherein said DNA virus is an
adenovirus.
12. The vector of claim 10, wherein said first coding sequence is
selected from the group consisting of the E1a coding sequence, and
the E1b coding sequence.
13. The vector of claim 11, wherein said second coding sequence
replaces a coding sequence nonessential for vector replication, so
that said second coding sequence is operably linked to a
transcriptional regulatory sequence from said gene nonessential for
vector replication.
14. The vector of claim 13, wherein said coding sequence
nonessential for vector replication is selected from the group
consisting of E3 coding sequences, E4 coding sequences, E1b coding
19 kD coding sequence, and E1b55 kD coding sequence.
15. A cell containing the vector of claim 1.
16. The cell of claim 15, wherein said vector replicates in said
cell by means of said tissue-specific regulatory sequence, and in
which cell said additional coding sequences are capable of
expression.
17. The cell of claim 15, wherein said cell is a tumor cell or an
abnormally proliferating cell.
18. The cell of claim 17, wherein said additional coding sequence
provides a gene product that provides anti-tumor activity in said
cell.
19. The cell of claim 17, wherein said tumor cell is selected from
the group consisting of a hepatoma cell, and lung carcinoma
cell.
20. A virion containing the vector of claim 1.
21. A cell containing the virion of claim 20, wherein said cell is
a producer cell for said virion.
22. A cell containing the virion of claim 20, wherein said
transcriptional regulatory sequence functions in said cell so that
replication of the virion and expression of the additional coding
sequence occurs in said cell.
23. A cell containing the virion of claim 20, wherein cell is a
tumor cell.
24. A cell containing the virion of claim 23, wherein said
heterologous gene product provides anti-tumor activity in said
cell.
25. A method of producing the vector of claim 1, comprising
culturing the cell of claim 15 and recovering said vector from said
cell.
26. A method of producing the virion of claim 20, comprising
culturing the cell of claim 21 and recovering said virion from said
cell.
27. A method for distributing a polynucleotide in a tissue in vivo,
comprising introducing said vector of claim 1 into said tissue and
allowing replication of said vector to occur in said tissue.
28. The vector of claim 1, wherein said additional coding sequence
expresses a gene product that can reduce or eliminate vector
replication.
29. The vector of claim 28, wherein said gene product is selected
from the group consisting of cytosine deaminase, thymidine kinase,
and purine nucleoside phosphorylase.
30. A method for modulating the replication of the vector of claim
28, comprising introducing a nucleoside analogue that is
phosphorylated by said gene product, when said vector is
replicating in a cell.
31. A method for expressing a gene in a cell, comprising
introducing the vector of claim 1 into a cell, and allowing
expression of said additional coding sequences in said cell.
32. The method of claim 30, wherein said cell is a tumor cell or an
abnormally proliferating cell.
33. A method for diagnosing a cell for the ability to replicate the
vector of claim 1 and express said heterologous gene product
therefrom, comprising introducing said vector into said cell and
assaying said cell for vector replication and gene expression.
34. A method for diagnosing a tumor for the ability to replicate
the vector of claim 1 and express said heterologous gene product
therefrom, and subsequently treating said tumor in a patient,
comprising (a) explanting a tumor biopsy from said patient, (b)
introducing into the cells of said biopsy the vector of claim 1,
(c) assaying said vector replication and expression in said cells,
and (d) introducing said vector into said patient.
35. The method of claim 34, further comprising (e) adding a
nucleoside analogue.
36. The method of claim 35, wherein said nucleoside analogue has
anti-tumor activity or eliminates cell proliferation.
37. The method of claim 35, wherein said nucleoside analogue is
selected from the group consisting of ganciclovir, acycolvir,
1,2-deoxy-2-fluoro-.beta.-D-arabinofuranosil-5-iodouracil, and
fencytovir.
38. A virion comprising a tissue-specific replication-conditional
adenoviral vector comprising: (a) a heterologous tissue-specific
transcriptional regulatory sequence operably linked to the coding
region of a gene that is essential for replication of said vector,
wherein said coding region is an E1a, E1b, E2, or E4 coding region;
and (b) at least one additional coding sequence encoding a
heterologous gene product, wherein said additional coding sequence
is operably linked to said heterologous tissue-specific
transcriptional regulatory sequence.
39. The virion of claim 38, wherein said tissue-specific
transcriptional regulatory sequence is a promoter or an
enhancer.
40. The virion of claim 39, where said promoter is selected from
the group consisting of an MUC1/DF3 promoter, an alpha-fetoprotein
promoter, an erb-B2 promoter, a surfactant promoter, a thymidine
kinase promoter, a p21 promoter, and a cyclin promoter.
41. The virion of claim 39, wherein said enhancer is selected from
the group consisting of DF3, a breast cancer-specific enhancer,
viral enhancers, and steroid receptor enhancers.
42. The virion of claim 38, wherein said additional coding sequence
is selected from the group consisting of a thymidine kinase coding
sequence, a cytosine deaminase coding sequence, and a purine
nucleoside phosphorylase coding sequence.
43. An isolated cell comprising the virion of claim 38.
44. An isolated cell comprising the virion of claim 38, wherein
said transcriptional regulatory sequence functions in said cell so
that replication of said virion and expression of said additional
coding sequence occurs in said cell.
45. The cell of claim 43, wherein said cell is a tumor cell or an
abnormally proliferating cell.
46. The cell of claim 45, wherein said additional coding sequence
provides a gene product that provides anti-tumor activity in said
cell.
47. The cell of claim 45, wherein said tumor cell is selected from
the group consisting of a hepatoma cell, and lung carcinoma
cell.
48. A method of producing the virion of claim 38, comprising
culturing a cell infected with said virion and recovering said
virion from said cell.
49. The virion of claim 38, wherein said additional coding sequence
expresses a gene product that can reduce or eliminate virion
replication.
50. The virion of claim 49, wherein said gene product is selected
from the group consisting of cytosine deaminase, thymidine kinase,
and purine nucleoside phosphorylase.
51. A virion comprising a tissue-specific replication-conditional
adenoviral vector comprising: (a) a heterologous tissue-specific
transcriptional regulatory sequence operably linked to the coding
region of the adenovirus E1a gene that is essential for replication
of said vector; and (b) at least one additional coding sequence
encoding a heterologous gene product, wherein said additional
coding sequence is operably linked to a second transcriptional
regulatory sequence that is activated by the E1a gene product.
52. The virion of claim 51, wherein said at least one additional
coding sequence replaces a coding sequence of a gene in said
vector, which gene is not essential for vector replication, such
that said at least one additional coding sequence is operably
linked to and transcribed from said second transcriptional
regulatory sequence.
53. The virion of claim 51, wherein at least one of said
transcriptional regulatory sequences is a promoter or an
enhancer.
54. The virion of claim 53, where said promoter is selected from
the group consisting of an MUC1/DF3 promoter, an alpha-fetoprotein
promoter, an erb-B2 promoter, a surfactant promoter, a thymidine
kinase promoter, a p21 promoter, and a cyclin promoter.
55. The virion of claim 53, wherein said enhancer is selected from
the group consisting of DF3, a breast cancer-specific enhancer, a
viral enhancer, and a steroid receptor enhancer.
56. The virion of claim 51, wherein said additional coding sequence
is selected from the group consisting of a thymidine kinase coding
sequence, a cytosine deaminase coding sequence, and a purine
nucleoside phosphorylase coding sequence.
57. The virion of claim 51, wherein said at least one additional
coding sequence encodes a gene product that can reduce or eliminate
replication of said vector.
58. The virion of claim 57, wherein said gene product is selected
from the group consisting of cytosine deaminase, thymidine kinase,
and purine nucleoside phosphorylase.
59. An isolated cell comprising the virion of claim 51.
60. An isolated cell comprising the virion of claim 51, wherein
said transcriptional regulatory sequence operably linked to the
coding region of the adenovirus E1a gene functions in said cell so
that replication of said virion and expression of said additional
coding sequence occurs in said cell.
61. The cell of claim 59, wherein said cell is a tumor cell or an
abnormally proliferating cell.
62. The cell of claim 61, wherein said at least one additional
coding sequence encodes a gene product that provides anti-tumor
activity in said cell.
63. The cell of claim 61, wherein said tumor cell is selected from
the group consisting of a hepatoma cell and lung carcinoma
cell.
64. A method of producing the virion of claim 51, comprising
culturing a cell infected with said vector and recovering said
vector from said cell.
65. The virion of claim 38, wherein said transcriptional regulatory
sequence is a tumor-specific regulatory sequence.
66. The virion of claim 65, wherein said tumor-specific regulatory
sequence is a tumor-specific promoter.
67. The virion of claim 38, wherein said transcriptional regulatory
sequence is an alpha-fetoprotein promoter.
68. The virion of claim 38, wherein said coding region is the E1a
coding region.
69. The virion of claim 38, wherein said coding region is the E1b
coding region.
70. The virion of claim 38, wherein said coding region is an E2
coding region.
71. The virion of claim 70, wherein said coding region is the E2a
coding region.
72. The virion of claim 38, wherein said coding region is the E4
coding region.
73. The virion of claim 38, wherein said additional coding sequence
is a thymidine kinase coding sequence.
74. The cell of claim 43, wherein said transcriptional regulatory
sequence is a tumor-specific regulatory sequence.
75. The cell of claim 74, wherein said tumor-specific regulatory
sequence is a tumor-specific promoter.
76. The cell of claim 43, wherein said transcriptional regulatory
sequence is an alpha-fetoprotein promoter.
77. The cell of claim 43, wherein said coding region is the E1a
coding region.
78. The cell of claim 43, wherein said coding region is the E1b
coding region.
79. The cell of claim 43, wherein said coding region is an E2
coding region.
80. The cell of claim 79, wherein said coding region is the E2a
coding region.
81. The cell of claim 43, wherein said coding region is the E4
coding region.
82. The cell of claim 43, wherein said additional coding sequence
is a thymidine kinase coding sequence.
83. The virion of claim 51, wherein said transcriptional regulatory
sequence operably linked to the coding region of the adenovirus E1a
gene is a tumor-specific regulatory sequence.
84. The virion of claim 83, wherein said tumor-specific regulatory
sequence operably linked to the coding region of the adenovirus E1a
gene is a tumor-specific promoter.
85. The virion of claim 51, wherein said transcriptional regulatory
sequence operably linked to the coding region of the adenovirus E1a
gene is an alpha-fetoprotein promoter.
86. The virion of claim 51, wherein said at least one additional
coding sequence replaces a coding sequence of the adenovirus E3
gene in said vector, such that said at least one additional coding
sequence is operably linked to and transcribed from said second
transcriptional regulatory sequence.
87. The virion of claim 86, wherein said second transcriptional
regulatory sequence is an adenovirus E3 promoter.
88. The virion of claim 51, wherein said additional coding sequence
is a thymidine kinase coding sequence.
89. The virion of claim 87, wherein said additional coding sequence
is a thymidine kinase coding sequence.
90. The cell of claim 59, wherein said transcriptional regulatory
sequence operably linked to the coding region of the adenovirus E1a
gene is a tumor-specific regulatory sequence.
91. The cell of claim 90, wherein said tumor-specific regulatory
sequence operably linked to the coding region of the adenovirus E1a
gene is a tumor-specific promoter.
92. The cell of claim 59, wherein said transcriptional regulatory
sequence operably linked to the coding region of the adenovirus E1a
gene is an alpha-fetoprotein promoter.
93. The cell of claim 59, wherein said at least one additional
coding sequence replaces a coding sequence of the adenovirus E3
gene in said vector, such that said at least one additional coding
sequence is operably linked to and transcribed from said second
transcriptional regulatory sequence.
94. The cell of claim 93, wherein said second transcriptional
regulatory sequence is an adenovirus E3 promoter.
95. The cell of claim 59, wherein said additional coding sequence
is a thymidine kinase coding sequence.
96. The cell of claim 94, wherein said additional coding sequence
is a thymidine kinase coding sequence.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a division of U.S. application Ser. No.
08/974,391, filed Nov. 19, 1997, which is a continuation-in-part of
U.S. application Ser. No. 08/487,992, filed Jun. 7, 1995
(abandoned), which is a continuation-in-part of U.S. application
Ser. No. 08/348,258, filed Nov. 28, 1994 (abandoned). Said U.S.
application Ser. No. 08/974,391, filed Nov. 19, 1997, is also a
continuation-in-part of U.S. application Ser. No. 08/849,117, filed
Jul. 1, 1997 (U.S. Pat. No. 5,998,205), which is a .sctn.371 of
PCT/US95/15455, which has an international filing date under the
PCT of Nov. 28, 1995, and which entered the United States national
phase on May 28, 1997, which is a continuation-in-part of U.S.
application Ser. No. 08/487,992, filed Jun. 7, 1995 (abandoned),
which is a continuation-in-part of U.S. application Ser. No.
08/348,258, filed Nov. 28, 1994 (abandoned), all incorporated
herein in their entirety by reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The invention generally relates to cell-specific expression
vectors. It particularly relates to targeted gene therapy using
recombinant expression vectors and particularly adenovirus vectors.
The invention specifically relates to modulatable
replication-conditional expression vectors and methods for using
them. Such vectors are able to selectively replicate in a target
cell or tissue to provide a therapeutic benefit in a tissue from
the presence of the vector per se or from one or more heterologous
gene products expressed from the vector and distributed throughout
the tissue, and which vectors are designed so that replication and
gene expression from the vector can be modulated.
[0004] In such vectors, a gene essential for replication is placed
under the control of a heterologous tissue-specific transcriptional
regulatory sequence. Thus, replication is conditioned on the
presence of a factor(s) that induces transcription or the absence
of a factor(s) that inhibits transcription of the gene by means of
the transcriptional regulatory sequence.
[0005] Preferred vectors contain a heterologous gene that produces
a product that increases or inhibits viral replication. Such genes
are useful for modulating viral replication and thus also for
modulating expression of genes in the vector. With these vectors,
therefore, the vector can be expressed in a desirable cell, target
tissue can be selectively treated, and replication and expression
modulated.
[0006] The invention also relates to cells and/or methods to
produce multiple heterologous gene products in high quantity from
essentially one promoter element.
[0007] The invention also relates to methods of using the vectors
to screen a tissue for the presence or absence of transcriptional
regulatory functions that permit vector replication by means of the
transcriptional regulatory sequence.
[0008] The invention also relates to cells for producing high
titers of the recombinant replication-conditional vector and/or
high titers of a gene product expressed from the vector, useful for
making clinically relevant gene products or vector preparations for
targeted gene therapy.
[0009] 2. Background Art
Targeting Vectors
[0010] The introduction of exogenous genes into cells in vitro or
in vivo, systemically or in situ, has been of limited use for
compositions in which it would be disadvantageous for non-target
cells to take up the exogenous gene. One strategy to overcome this
problem is to develop administration procedures or vectors that
target a specific cell-type. Using systemic administration,
attempts have been made to direct exogenous genes to myocytes and
muscle cells by direct injection of DNA, to direct the exogenous
DNA to hepatocytes using DNA-protein complexes, and to endothelial
cells using liposomes.
[0011] Using in situ administration, retroviral replication
functions have been utilized to target cells that are actively
replicating.
[0012] Thus far, the ability to target cells has been limited,
however, by the lack of cell-type specificity and low gene transfer
efficiencies. The limited ability to target an exogenous gene to
diseased cells in an organism, while avoiding (eliminating) uptake
of the gene by normal, untargeted cells, particularly has been an
obstacle to developing effective gene-transfer-based therapies for
diseases in animals and humans.
[0013] One especially difficult challenge is targeting tumor cells.
Many seemingly promising strategies for these cells, moreover, are
limited to one or a few cell-types.
[0014] The present invention, in one aspect, provides a way to
deliver an exogenous gene efficiently, with high distribution in a
tumor and in a controlled manner.
Adenoviruses Generally
[0015] Adenoviruses are nonenveloped, regular icosohedrons. The
protein coat (capsid) is composed of 252 capsomeres of which 240
are hexons and 12 are pentons. Most of the detailed structural
studies of the adenovirus polypeptides have been done for
adenovirus types 2 and 5. The viral DNA is 23.85.times.10.sup.6
daltons for adenovirus 2 and varies slightly in size depending on
serotype. The DNA has inverted terminal repeats and the length of
these varies with the serotype.
[0016] The replicative cycle is divided into early (E) and late (L)
phases. The late phase defines the onset of viral DNA replication.
Adenovirus structural proteins are generally synthesized during the
late phase. Following adenovirus infection, host DNA and protein
synthesis is inhibited in cells infected with most serotypes. The
adenovirus lytic cycle with adenovirus 2 and adenovirus 5 is very
efficient and results in approximately 10,000 virions per infected
cell along with the synthesis of excess viral protein and DNA that
is not incorporated into the virion. Early adenovirus transcription
is a complicated sequence of interrelated biochemical events, but
it entails essentially the synthesis of viral RNAs prior to the
onset of viral DNA replication.
[0017] The organization of the adenovirus genome is similar in all
of the adenovirus groups and specific functions are generally
positioned at identical locations for each serotype studied. Early
cytoplasmic messenger RNAs are complementary to four defined,
noncontiguous regions on the viral DNA. These regions are
designated (E1-E4). The early transcripts have been classified into
an array of immediate early (E1a), delayed early (E1b, E2a, E2b, E3
and E4), and intermediate (IVa2.IX) regions.
[0018] E1 a is a transactivator of multiple gene products in
adenovirus through activation of the E1b, E2, E3 and E4 promoters.
The E1a region is involved in transcriptional transactivation of
viral and cellular genes as well as transcriptional repression of
other sequences. The E1 a gene exerts an important control function
on all of the other early adenovirus messenger RNAs. In normal
tissues, in order to transcribe regions E1b, E2a, E2b, E3, or E4
efficiently, active E1a product is required.
[0019] The E1b region is required for the normal progression of
viral events late in infection. The E1b product acts in the host
nucleus. Mutants generated within the E1b sequences exhibit
diminished late viral mRNA accumulation as well as impairment in
the inhibition of host cellular transport normally observed late in
adenovirus infection (Berkner, K. L., Biotechniques 6:616-629
(1988)). E1b is required for altering functions of the host cell
such that processing and transport are shifted in favor of viral
late gene products. These products then result in viral packaging
and release of virions. E1b produces a 19 kD protein that prevents
apoptosis. E1b also produces a 55 kD protein that binds to p53.
[0020] For a complete review on adenoviruses and their replication,
see Horwitz, M. S., Virology 2d ed., Fields, B. N., eds., Raven
Press Limited, New York (1990), Chapter 60, pp. 1679-1721.
Adenovirus as Recombinant Delivery Vehicle
[0021] Adenovirus provides advantages as a vector for adequate gene
delivery for the following reasons. It is a double stranded DNA
nonenveloped virus with tropism for the human respiratory system
and gastrointestinal tract. It causes a mild flu-like disease.
Adenoviral vectors enter cells by receptor mediated endocytosis.
The large (36 kilobase) genome allows for the removal of genes
essential for replication and nonessential regions so that foreign
DNA may be inserted and expressed from the viral genome.
Adenoviruses infect a wide variety of cell types in vivo and in
vitro. Adenoviruses have been used as vectors for gene therapy and
for expression of heterologous genes. The expression of viral or
foreign genes from the adenovirus genome does not require a
replicating cell. Adenovirus vectors rarely integrate into the host
chromosome; the adenovirus genome remains as an extrachromosomal
element in the cellular nucleus. There is no association of human
malignancy with adenovirus infection; attenuated strains have been
developed and have been used in humans for live vaccines.
[0022] For a more detailed discussion of the use of adenovirus
vectors for gene therapy, see Berkner, K. L., Biotechniques
6:616-629 (1988); Trapnell, B. C., Advanced Drug Delivery Reviews
12: 185-199 (1993).
[0023] Adenovirus vectors are generally deleted in the E1 region of
the virus. The E1 region may then be substituted with the DNA
sequences of interest. It was pointed out in a recent article on
human gene therapy, however, that "the main disadvantage in the use
of adenovirus as a gene transfer vector is that the viral vector
generally remains episomal and does not replicate, thus, cell
division leads to the eventual loss of the vector from the daughter
cells" (Morgan, R. A., et al., Annual Review of Biochemistry
62:191-217 (1993)) (emphasis added).
[0024] Non-replication of the vector leads not only to eventual
loss of the vector without expression in most or all of the target
cells but also leads to insufficient expression in the cells that
do take up the vector, because copies of the gene whose expression
is desired are insufficient for maximum effect. The insufficiency
of gene expression is a general limitation of all non-replicating
delivery vectors. Thus, it is desirable to introduce a vector that
can provide multiple copies of a gene and hence greater amounts of
the product of that gene. The present invention overcomes the
disadvantages discussed above by providing a tissue-specific, and
especially a tumor-specific replicating vector, multiple DNA
copies, and thus increased amounts of gene product.
Production of Adenoviral Vectors
[0025] Adenoviral vectors for recombinant gene expression have been
produced in the human embryonic kidney cell line 293 (Graham, F. L.
et al., J. Gen. Virol. 36:59-72 (1977)). This cell line, initially
transformed with human adenovirus 5, now contains the left end of
the adenovirus 5 genome and expresses E1. Therefore, these cells
are permissive for growth of adenovirus 2 and adenovirus 5 mutants
defective in E1 functions. They have been extensively used for the
isolation and propagation of E1 mutants. Therefore, 293 cells have
been used for helper-independent cloning and expression of
adenovirus vectors in mammalian cells. E1 genes integrated in
cellular DNA of 293 cells are expressed at levels which permit
deletion of these genes from the viral vector genome. The deletion
provides a nonessential region into which DNA may be inserted. For
a review, see, Young, C. S. H., et al. in The Adenovintses,
Ginsberg, H. S., ed., Plenum Press, New York and London (1984), pp.
125-172.
[0026] However, 293 cells are subject to severe limitations as
producer cells for adenovirus vectors. Growth rates are low. Titres
are limited, especially when the vector produces a heterologous
gene product that proves toxic for the cells. Recombination with
the viral E1 sequence in the genome can lead to the contamination
of the recombinant defective virus with unsafe wild-type virus. The
quality of certain viral preparations is poor with regard to the
ratio of virus particle to plaque forming unit. Further, the cell
line does not support growth of more highly deleted mutants because
the expression of E1 in combination with other viral genes in the
cellular genome (required to complement the further deletion), such
as E4, is toxic to the cells. Therefore, the amount of heterologous
DNA that can be inserted into the viral genome is limited in these
cells. It is desirable, therefore, to produce adenovirus vectors
for gene therapy in a cell that cannot produce wild-type
recombinants and can produce high titres of high-quality virus.
[0027] It is also desirable to control the replication of a
therapeutic vector by other than endogenous cellular factors
present within the cells to be treated. A high degree of
replication could be disadvantageous to the patient, in that vector
particles could be released into the bloodstream. This could have
toxic side effects in tissues clearing the virus, such as kidneys,
liver, lung and spleen, even if the vector does not replicate in
non-treated tissue. Furthermore, while current therapeutic vectors
have not been shown to replicate at low exposure to normal cells, a
high exposure, as potentially caused from a high degree of
replication and release of virus at the site of the treated tissue,
may cause replication even in normal cells. For example,
adenoviruses devoid of E1a, given in a sufficiently high dose, will
replicate in normal cells.
[0028] Accordingly, it would be highly advantageous to be able to
control the level of replication by adding a compound that could
dampen replication if replication were excessive.
[0029] Another problem in the art is providing more than one gene
product controlled by a heterologous regulatory sequence, for
example a promoter, on a viral vector (such as cytokines, TK, or
other cytotoxic genes, or heat-shock proteins). Thus, in a specific
milieu, a particular combination of genes may offer therapeutic
advantage. Accordingly, it would be desirable to have several genes
in one vector and specifically expressed in the cells to be
treated, for example, in a tumor cell. A limitation, however, is
that one cannot provide multiple copies of the same promoter to
drive each gene, because space would be wasted and the excess
nucleotide stretches might not even be accommodated by the virus.
Furthermore, during the cloning and replication, homologous
recombination could produce deletions between identical promoters
and therefore destroy the vector.
SUMMARY OF THE INVENTION
[0030] In view of the limitations discussed above, a general object
of the invention is to provide novel expression vectors for
tissue-specific vector replication and gene expression from the
replicating vector.
[0031] Accordingly, the invention is directed to an expression
vector that contains at least one gene that is essential for
replication, which gene is operably linked to a heterologous
transcriptional regulatory sequence (heterologous with respect to
the gene essential for replication and/or to the vector type), such
that an expression vector is created whose replication is
conditioned upon the presence of a trans-acting transcriptional
regulatory factor(s) that permits transcription from the
transcriptional regulatory sequence, or the absence of a
transcriptional regulatory factor(s) that normally prevents
transcription from that transcriptional regulatory sequence. Thus,
these regulatory sequences are specifically activated or
derepressed in the target cell or tissue so that replication of the
vector proceeds in that cell or tissue and expression of
heterologous genes is induced or amplified.
[0032] Another object of the invention is to provide an expression
vector whose replication, and hence gene expression, can be
modulated.
[0033] Accordingly, a vector is provided that contains a gene
encoding a gene product that can affect the rate and extent of
vector replication.
[0034] Another object of the invention is to provide a way to
coordinate and amplify the expression of multiple genes on a single
vector.
[0035] Accordingly, an expression vector is provided in which the
expression of multiple genes on the vector can be controlled and
coordinated through the expression of a gene that is essential for
replication, so that the expression of each of the multiple genes
is conditional upon vector replication, replication depending upon
the tissue-specific expression of the gene product.
[0036] Another object of the invention is to provide
tissue-specific treatment of abnormal tissue. Thus, a further
object of the invention is to provide a method to selectively
distribute a vector in vivo in a target tissue, such that a greater
number of cells contain the vector than would with a
non-replicating vector, and spread of the vector is avoided or
significantly reduced in non-target tissue.
[0037] Accordingly, a method is provided for selectively
distributing a vector in a target tissue by introducing the
replication-conditional vector of the present invention into a
target tissue that allows modulatable replication of the
vector.
[0038] For providing tissue-specific treatment, another object of
the invention is to selectively distribute a polynucleotide in a
target tissue in vivo.
[0039] Accordingly, the invention is directed to a method for
selectively distributing a polynucleotide in a target tissue in
vivo by introducing the replication-conditional vectors of the
present invention, containing the polynucleotide, into the target
tissue that allows modulatable replication of the vector. The
polynucleotide includes the entire vector or parts thereof, such as
one or more heterologous genes.
[0040] For providing tissue-specific treatment, a further object of
the invention is to selectively distribute one or more heterologous
gene products in a target tissue.
[0041] Accordingly, the replication-conditional vectors of the
present invention are constructed so that they contain one or more
heterologous DNA sequences encoding a gene product that is
expressed from the vector. When the vectors replicate in the target
tissue, effective quantities of the desired gene product are also
produced in the target tissue.
[0042] Another object of the invention, especially where
tissue-specific treatment is involved, is to be able to modulate
vector replication, that is, control the level of vector
replication. When vector replication proceeds at levels that are
undesirable, and particularly levels that may interfere with
treatment, an object of the invention is to dampen or decrease the
levels of replication by a desired degree. If the levels fall below
the desirable amount, an object of the invention is also to be able
to allow replication to increase to desirable levels by desired
degrees.
[0043] Accordingly, the invention provides a method to modulate
vector replication during treatment or otherwise (e.g., in producer
cells), by providing a gene encoding a gene product that has the
ability to interfere with vector replication. When replication is
undesirably high, it can be decreased by means of this gene
product.
[0044] Thus, the invention is further directed to vectors further
containing a gene encoding a gene product that can be used to
modulate vector replication.
[0045] Modulating replication also modulates the expression of
heterologous genes contained in the vector. Thus, the expression of
such genes can be induced, decreased, increased, or eliminated
using the methods and vectors described herein.
[0046] Another object of the invention is to provide a method to
identify abnormal tissue that can then be treated by the vectors of
the present invention. Therefore, a further object of the invention
is to identify a tissue in which the replication-conditional
vectors of the present invention can be replicated by means of the
heterologous transcriptional regulatory sequence contained on the
vector so that the tissue can subsequently be treated with the
vector, and in which tissue the replication can be modulated.
[0047] Accordingly, the invention is further directed to a method
wherein the replication-conditional vectors of the present
invention are exposed to a given abnormal tissue. If that tissue
allows replication and modulation, then replication of the vector
will occur and can be detected. Following identification of such a
tissue, targeted treatment of that tissue can be effected by
tissue-specific transcription and the consequent vector replication
in that tissue in vivo.
[0048] Thus, a method is provided for assaying vector utility for
tissue treatment comprising the steps of removing a tissue biopsy
from a patient, explanting the biopsy into tissue culture,
introducing a replication-conditional vector into the cells of the
biopsy, and assaying for modulatable vector replication in the
cells.
[0049] Another object of the invention is to provide producer cell
lines for vector production. Preferably, the cell lines have one or
more of the following characteristics: high titer virus production,
resistance to toxic effects due to heterologous gene products
expressed in the vector, lack of production of wild-type virus
contaminating the virus preparation and resulting from
recombination between integrated viral sequences and vector
sequences, growth to high density and in suspension, unlimited
passage potential, high growth rate, and by permitting the growth
of highly deleted viruses that are impaired for viral functions and
able to accommodate large pieces of heterologous DNA.
[0050] Accordingly, in a further embodiment of the invention, cell
lines are provided containing the replication-conditional vector of
the present invention, the cells of which allow modulatable
replication of the vector or is deficient in a
transcription-inhibiting factor(s) that prevents replication of the
vector.
[0051] In further embodiments of the invention, the cell lines
contain nucleic acid copies of the replicated vector. In other
embodiments, the cell lines contain virions produced in the cell by
replication in the cell of the replication-conditional vector.
[0052] In further embodiments, a method is provided for producing a
replication-conditional vector or virion comprising the steps of
culturing a producer cell line described above and recovering the
vector or virion from the cells.
[0053] In still further embodiments, a method is provided for
producing replication-conditional virions free of wild-type virions
or viral vectors free of wild-type vectors, comprising the steps of
culturing a producer cell line described above and recovering the
replication-deficient virions or vectors from the cells.
[0054] In a preferred methods of treatment and diagnosis, the
tissue is abnormally proliferating, and especially is tumor tissue.
However, the methods are also directed to other abnormal tissue as
described herein.
[0055] In a preferred embodiment of the invention, the
replication-conditional vector is a DNA tumor viral vector.
[0056] In a further preferred embodiment, the DNA tumor viral
vector is a vector selected from the group consisting of
herpesvirus, papovavirus, papillomavirus, parvovirus and hepatitis
virus vectors.
[0057] In a most preferred embodiment, the vector is an adenovirus
vector.
[0058] However, it is to be understood that potentially any vector
source is useful if it contains a gene essential for replication
that can be operably linked to a tissue-specific transcriptional
regulatory sequence.
[0059] In further methods of treatment and diagnosis, the vector is
introduced into the cell or tissue by infection.
[0060] Replication can be vector nucleic acid replication alone or
can also include virus replication (i.e., virion production). Thus,
either DNA or virions or both may be distributed in the target
tissue.
[0061] In a further preferred embodiment of the invention, a gene
in the adenovirus E1 region is operably linked to the
tissue-specific heterologous transcriptional regulatory sequence.
Preferably, the E1a, E1b or E2a gene is operably linked to the
tissue-specific transcriptional regulatory sequence.
[0062] In a further embodiment of the invention, the vector encodes
one or more heterologous gene products. These heterologous gene
products are expressed from the vector replicating in the target
tissue. The heterologous gene product may be operably linked to its
own promoter or may be operably linked to a transcriptional
regulatory sequence from another gene. Regardless, the expression
of the heterologous gene product may be controlled by E1a or
another transactivator which in turn is regulated by the desired
tumor-specific promoter.
[0063] In preferred embodiments of the invention, expression of one
or more heterologous gene products depends upon expression of the
gene essential for replication, which in turn is operably linked to
the tissue-specific transcriptional regulatory sequence.
Accordingly, when the gene essential for replication is expressed,
it causes expression of one or more heterologous gene products, for
example by transactivation. In this configuration, such a
heterologous gene is activated/induced only when the vector
replicates. This is because activation of the tissue-specific
regulatory sequence causes expression of the gene product essential
for replication which then causes both replication and activation
of the expression of the one or more heterologous genes by its
transactivation function. Accordingly, not only is expression of
the heterologous gene activated, but expression is also amplified
because when the vector replicates, each additional copy of the
vector also contains a copy of the heterologous gene.
[0064] In a highly preferred embodiment, the E1a gene, being
operably linked to a heterologous tissue-specific transcriptional
regulatory sequence, controls the expression of one or more
heterologous genes under the control of promoters that are
transactivated by the E1a gene product. Thus, when the E1a gene is
expressed, viral replication occurs and gene expression of one or
more heterologous genes also occurs. Thus, expression of those
genes is controlled at the level of replication and
transactivation, such that an expression-amplifying effect is
obtained.
[0065] In a further embodiment, one or more of the heterologous
genes is operably linked to a tissue-specific regulatory sequence,
such as the same transcriptional regulatory sequence to which the
gene essential for replication is operably linked. Accordingly, the
one or more heterologous genes are then activated only in a
specific tissue. When the tissue-specific transcriptional
regulatory sequence is the same one as that to which the gene
essential for replication is operably linked, activation of the one
or more heterologous genes occurs only when the vector replicates.
Accordingly, as above, the one or more heterologous genes is both
activated and amplified.
[0066] In other embodiments, the one or more heterologous genes is
amplified when the vector replicates but not necessarily activated.
This is when these genes are under the control of their own or
other constitutive promoters. In this case, there is always a basal
level of expression, but when the vector replicates, this
expression is amplified because of expression from each new copy of
the vector.
[0067] In the context of coordinate control (one or more
heterologous genes under the control of the same transcriptional
regulatory sequence, the same scenario of activation and
amplification as discussed above applies.
[0068] In a further embodiment, the vector is not used to
selectively destroy a cell or tissue type, such as a tumor, but is
used to provide a gene product or products at specific levels that
are physiologically desirable. An example of such gene product is
insulin. Thus, using replication to control gene expression in
order to achieve a very specific amount of gene expression is
provided by the invention. Accordingly, the vector encodes a gene
product which regulates the degree of virus replication, and hence
gene expression of other gene products whose amounts must be
controlled. An example of such product is one that would inhibit
DNA polymerase or nucleotide synthesis. Accordingly, an excess of
this product will down-regulate the vector replication. Gene
products are also encompassed that will increase the level of
vector replication if sufficient gene product is not produced.
[0069] Thus, according to the invention, one or more of the
heterologous gene products is useful for modulating viral
replication. It may directly or indirectly control viral
replication as, for example, in the case of thymidine kinase in
which viral replication is negatively affected and modulated by the
addition of ganciclovir.
[0070] With respect to modulating gene expression, the vector
provides for modulating the expression of heterologous genes in any
of the configurations described above by modulating replication of
the vector. This is preferably done via thymidine kinase or a gene
product functioning as thymidine kinase does.
[0071] In a highly preferred embodiment, the thymidine kinase
coding region is operably linked to the E3 promoter in an
adenoviral vector. The E1a coding region is under the control of a
heterologous tissue-specific promoter. Accordingly, when the
tissue-specific promoter is activated, the E1a gene product is
produced. This gene product then transactivates the E3 promoter, so
that the TK gene is expressed. Further, since the E1a gene
activates viral replication, and the E3 gene is not essential for
replication, more copies of the viral vector are present to allow
greater expression of the TK gene. Ganciclovir can then be added to
modulate vector replication. If after adding the ganciclovir,
replication falls below a desirable level, the amount of
ganciclovir can be decreased to allow an increase in vector
replication and so forth, so that desirable levels are permitted
within any particular time frame. One example of desirable
modulation is to decrease vector replication in a non-target
cell.
[0072] In a further embodiment of the invention, a heterologous
gene product is toxic for the target tissue.
[0073] In a further embodiment of the invention, the toxic
heterologous gene product acts on a non-toxic prodrug, converting
the non-toxic prodrug into a form that is toxic for the target
tissue. Preferably, the toxin has anti-tumor activity or eliminates
cell proliferation.
[0074] In preferred embodiments of the invention, the
transcriptional regulatory sequence is a promoter. Preferred
promoters include, but are not limited to, carcinoembryonic antigen
(CEA), DF3, .alpha.-fetoprotein (AFP), Erb-B2, surfactant, and
especially lung surfactant, tyrosinase promoter, and
endothelial-specific promoters. However, any genetic control region
that controls transcription of the essential gene can be used to
activate (or derepress) the gene. Thus, other genetic control
elements, such as enhancers, repressible sequences, and silencers,
can be used to regulate replication of the vector in the target
cell. The only requirement is that the genetic element be
activated, derepressed, enhanced, or otherwise genetically
regulated by factors in the host cell and, with respect to methods
of treatment, not in the non-target cell.
[0075] Preferred enhancers include the DF3 breast cancer-specific
enhancer and enhancers from viruses and the steroid receptor
family. Other preferred transcriptional regulatory sequences
include NF1, SP1, AP1, and FOS/JUN.
[0076] In further embodiments, promoters are not necessarily
activated by factors in the target tissue, but are derepressed by
factors present in the target tissue. Thus, in the target tissue,
repression is lifted.
[0077] Transcriptional regulatory factors include, but are not
limited to, transactivating factors produced by endogenous viral
sequences such as from cytomegalovirus (CMV), HIV, Epstein-Barr
virus (EBV), Herpes simplex virus (HSV), SV40, and other such
viruses that are pathogenic in mammals and, particularly, in
humans.
[0078] Methods for making such vectors are well known to the person
of ordinary skill in the art. The art adequately teaches the
construction of recombinant vectors with deletions or modifications
in specific coding sequences and operable linkage to a heterologous
transcription control sequence such that expression of a desired
coding region is under control of the heterologous transcriptional
regulatory sequence. Many viral sequences have been adequately
mapped such that it is routine to identify a gene of choice and use
appropriate well known techniques (such as homologous recombination
of the virus with deleted or otherwise modified plasmids, or
ligation of the two) to construct the vectors for tissue-specific
replication and expression.
BRIEF DESCRIPTION OF THE FIGURES
[0079] FIG. 1A. Cloning of pAVE1a02i.
[0080] FIG. 1B. Diagram of pAF(AB).sub.2(S).sub.6CAT. The map shows
the enhancer regions (AB) in opposite orientation.
[0081] FIG. 1C. Diagram of pAvE1a04i as predicted from a plasmid
map and sequence information describing pAF(AB).sub.2(S).sub.6CAT,
and pAvE1a06i as determined by restriction digestion mapping and
sequencing to be the correct structure.
[0082] FIG. 2A. Diagram of ClaI digestion of Av1LacZ.
[0083] FIG. 2B. Diagram of co-transfection of 293 cells with
pAvE1a06i and Av1LacZ ClaI digest (30,884 bp), to produce
AvE1a06i.
[0084] FIG. 2C. Amplification of AvE1a06i virus on S8 cells deleted
one direct enhancer (AB) from the promoter. The new construct is
designated AvE1a 04i.
[0085] FIG. 3A. Cloning of the plasmid pREpacTK.
[0086] FIG. 3B. Cloning of the plasmid pREpacTKm.
[0087] FIG. 3C. Construction of AV5E1aTK01i.
[0088] FIG. 4A. Digestion of Av1E1a04i.
[0089] FIG. 4B. Digestion of Av5E1aTK01i.
[0090] FIG. 4C. Construction of Av15E1aTK04i using the digestion
products of FIGS. 4A and 4B.
[0091] FIG. 5. Cytopathic effect of Av15E1aTK04i.
[0092] FIG. 6. GCV Inhibition of Av5E1aTk01i Replication In Vitro.
A549 cells were transduced with either Add1327 or Add1327Tk01i.
Cells were then treated with 10 uM GCV for 5 days. Following the 5
day incubation, cells were harvested, washed in HBSS, and then
freeze/thawed to prepare a crude viral lysate. Titers were then
performed by standard TCID50 assays on 293 cells to determine viral
titer. The experiment was repeated three times. Results show that
while GCV had no effect on an adenovirus not carrying the HSV-TK
gene, it caused a greater than 4 order of magnitude drop in titer
of a vector carrying the HSV-TK gene.
[0093] FIG. 7. GCV Inhibition of Av5E1aTk01i Replication In Vivo.
Subcutaneous tumors were formed by injecting 1.times.10.sup.7 A549
cells into the subcutaneous space of the right flank of nude mice.
After tumors formed 1.times.10.sup.9pfu of Add1327Tk01i was
injected into several animals containing tumors. After 5 days, half
of the animals received 5 days of IP GCV treatment at 75 mg/kg once
a day. At the end of this time frame all animals were sacrificed.
Immunohistochemistry was performed on all samples for HSV-TK
expression and hexon expression, the latter which is only expressed
when the vector is replicating. Results show as seen in the figure
that only hexon is severely diminished when the animals were
treated with GCV.
[0094] FIG. 8. Diagram of Possible Configurations of Heterologous
Genes in Expression Vectors.
[0095] FIGS. 9A-C. PCR identification of recombinant adenovirus
with E1a expressed from the hepatoma-specific AFP promoter. FIG. 9A
shows that viral plaques are produced by viral genomes containing
the AFP promoter operably linked to E1a. FIG. 9B shows that there
was no contamination with wild-type virus. FIG. 9C shows that there
was no contamination with AV1lacZ DNA.
[0096] FIGS. 10A-F. Tissue specific adenovirus with E1a expressed
from the AFP promoter. The experiment shows cytopathic effects and
spreading of cell death following infection with the virus
AVAFPE1a. FIGS. 10A-10C show uninfected controls in A549.30, A549,
and HuH 7 cells, respectively. FIGS. 10D-10F show the results of
infection with the virus in A549.30, A549, and HuH 7 cells,
respectively.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Definitions
[0097] The term "abnormally proliferating" is intended to mean a
cell having a higher mitotic index than its normally-functioning
counterpart, such that there is an abnormal accumulation of such
cells.
[0098] The term "anti-tumor activity" is intended to mean any
activity which inhibits, prevents, or destroys the growth of a
tumor.
[0099] As used herein, the term "cytotoxic gene" refers to a gene
that encodes a protein which either alone or in combination with
other agents is lethal to cell viability. Examples of cytotoxic
genes which alone are lethal include toxins such as pertussis
toxin, diphtheria toxin and the like.
[0100] Examples of cytotoxic genes which are used in combination
with other agents to achieve cell lethality include, for example,
herpes simplex-1 thymidine kinase and cytosine deaminase. The
subject is then administered an effective amount of a therapeutic
agent, which in the presence of the cytotoxic gene is toxic to the
cell. In the specific case of thymidine kinase, the therapeutic
agent is a thymidine kinase substrate such as ganciclovir (GCV),
6-methoxypurine arabinonucleoside (araM), or a functional
equivalent thereof. Both the thymidine kinase gene and the
thymidine kinase metabolite must be used concurrently to be toxic
to the host cell. However, in its presence, GCV is phosphorylated
and becomes a potent inhibitor of DNA synthesis whereas araM gets
converted to the cytotoxic anabolite araATP. Other genes can be
used as well in combination with the corresponding therapeutic
agent. Such other gene and therapeutic agent combinations are known
by one skilled in the art. Another example would be the vector of
this invention expressing the enzyme cytosine deaminase. Such
vector would be used in conjunction with administration of the drug
5-fluorouracil (Austin and Huber, 1993), or the recently described
E. coli Deo.DELTA. gene in combination with
6-methyl-purine-2'-deosribonucleoside (Sorscher et al., 1994).
[0101] The term "distributing" is intended to mean the spreading of
a vector and its attendant heterologous gene (product) (when
present on the vector) throughout a target tissue, and especially
throughout abnormally proliferating tissue (non-malignant or
malignant). The object of the distribution is to deliver the
vector, gene product or the effects of the gene product (as by a
bystander effect, for example) to substantially all or a
significant number of cells of the target tissue, so as to treat
substantially the entire target tissue.
[0102] The term "enhancer" is used according to its art-recognized
meaning. It is intended to mean a sequence found in eukaryotes and
certain eukaryotic viruses which can increase transcription from a
gene when located (in either orientation) up to several kilobases
from the gene being studied. These sequences usually act as
enhancers when on the 5' side (upstream) of the gene in question.
However, some enhancers are active when placed on the 3' side
(downstream) of the gene. In some cases, enhancer elements can
activate transcription from a gene with no (known) promoter.
[0103] The term "functional inactivation" is intended to mean a
genetic lesion that prevents the normal activity of a gene product.
Thus, functional inactivation could result from a mutation in the
gene encoding the gene product. Such a lesion includes insertions,
deletions, and base changes. Alternatively, functional inactivation
may occur by the abnormal interaction of the normal gene product
with one or more other cellular gene products which bind to or
otherwise prevent the functional activity of said gene product.
Thus, the gene product may be a protein produced from a normal gene
but which cannot perform its ordinary and normal function because
of an interaction with a second factor.
[0104] The term "gene essential for replication" refers to a
genetic sequence whose transcription is required for the vector to
replicate in the target cell.
[0105] The term "gene product" is intended to mean DNA, RNA,
protein, peptides, or viral particles. Thus, the distribution, for
the purposes of the invention, is of any of these components.
[0106] The term "heterologous" means a DNA sequence not found in
the native vector genome. Thus, for example, when the vector is
based on the adenovirus genome, heterologous DNA sequences are
those that are not found in the native adenovirus genome. With
respect to a "heterologous transcriptional regulatory sequence,"
"heterologous" indicates that the transcriptional regulatory
sequence is not naturally ligated to the DNA sequence for the gene
essential for replication of the vector.
[0107] The term "promoter" is used according to its art-recognized
meaning. It is intended to mean the DNA region, usually upstream to
the coding sequence of a gene or operon, which binds RNA polymerase
and directs the enzyme to the correct transcriptional start
site.
[0108] The term "replication" means duplication of a vector. This
duplication, in the case of viruses, can occur at the level of
nucleic acid, or at the level of infectious viral particle. In the
case of DNA viruses, replication at the nucleic acid level is DNA
replication. In the case of RNA viruses, nucleic acid replication
is replication into plus or minus strand (or both). In the case of
retroviruses, replication at the nucleic acid level includes the
production of cDNA as well as the further production of RNA viral
genomes. The essential feature is nucleic acid copies of the
original viral vector. However, replication also includes the
formation of infectious DNA or RNA viral particles. Such particles
may successively infect cells in a given target tissue thus
distributing the vector through all or a significant portion of the
target tissue.
[0109] The term "replication-conditional vector" refers to a vector
which when introduced into a tissue will not replicate unless a
transcriptional regulatory sequence in that vector is activated or
derepressed in that tissue. That is, replication depends upon
transcription by means of that transcriptional regulatory sequence.
Such a vector is replication-conditional as described because it
has been modified in the following manner. A gene that is essential
for replication has been modified by replacing the transcriptional
regulatory sequence on which transcription of that gene normally
depends with a heterologous transcriptional regulatory sequence.
This transcriptional regulatory sequence depends upon the presence
of transcriptional regulatory factors or the absence of
transcriptional regulatory inhibitors. The presence of these
factors in a given tissue or the absence of such inhibitors in a
given tissue provides the replication-conditionality. Accordingly,
the native transcriptional regulatory sequence may be replaced with
the heterologous transcriptional regulatory sequence.
Alternatively, the native transcriptional regulatory sequence may
be disabled or rendered dysfunctional by partial removal (deletion)
or other mutation (one or more base changes, insertions,
inversions, etc.).
[0110] The gene sequence may be a coding sequence. It may contain
one or more open reading frames, as well as intron sequences.
However, such a sequence is not limited to a coding sequence, but
includes sequences that are transcribed into RNA, which RNA is
itself essential for vector replication. The essential feature is
that the transcription of the gene sequences does not depend on the
native transcriptional regulatory sequences.
[0111] The term "silencer," used in its art-recognized sense, means
a sequence found in eucaryotic viruses and eucaryotes which can
decrease or silence transcription of a gene when located within
several kilobases of that gene.
[0112] The term "tissue-specific" is intended to mean that the
transcriptional regulatory sequence to which the gene essential for
replication is operably linked functions specifically in that
tissue so that replication proceeds in that tissue. This can occur
by the presence in that tissue, and absence in non-target tissues,
of positive transcription factors that activate the transcriptional
regulatory sequence. It can also occur by the absence of
transcription inhibiting factors that normally occur in non-target
tissues and prevent transcription as a result of the transcription
regulatory sequence. Thus, when transcription occurs, it proceeds
into the gene essential for replication such that in that target
tissue, replication of the vector and its attendant functions
occur.
[0113] As described herein, tissue specificity is particularly
relevant in the treatment of the abnormal counterpart of a normal
tissue. Such counterparts include, but are not limited to, liver
tissue and liver cancer, breast tissue and breast cancer, melanoma
and normal skin tissue. Tissue specificity also includes the
presence of an abnormal tissue type interspersed with normal tissue
of a different tissue type, as for example in the case of
metastases of colon cancer, breast cancer, and the like, into
tissue such as liver. In this case, the target tissue is the
abnormal tissue, and tissue specificity reflects the restriction of
vector replication to the abnormal tissue interspersed in the
normal tissue. It is also to be understood that tissue specificity,
in the context of treatment, is particularly relevant in vivo.
However, as described herein, ex vivo treatment and tissue
replacement also falls within the concept of tissue specificity
according to the present invention.
[0114] The term "transcriptional regulatory function" or
"transcriptional regulatory factor" is intended to mean any
cellular function whose presence activates or represses the
heterologous transcriptional regulatory sequence described herein
or whose absence permits transcription as a result of the
transcriptional regulatory sequences described herein. It is
understood that in the given target tissue, a tissue that "lacks
the transcriptional regulatory factor" or is "deficient" in the
transcriptional regulatory factor could refer to either the absence
of the factor or the functional inactivation of the factor in the
target tissue.
[0115] The term "transcriptional regulatory sequence" is used
according to its art-recognized meaning. It is intended to mean any
DNA sequence which can, by virtue of its sequence, cause the linked
gene to be either up- or down-regulated in a particular cell. In
one embodiment of the present invention, the native transcriptional
regulatory sequence is completely deleted from the vector and
replaced with a heterologous transcriptional regulatory sequence.
The transcriptional regulatory sequence may be adjacent to the
coding region for the gene that is essential for replication, or
may be removed from it. Accordingly, in the case of a promoter, the
promoter will generally be adjacent to the coding region. In the
case of an enhancer, however, an enhancer can be found at some
distance from the coding region such that there is an intervening
DNA sequence between the enhancer and the coding region. In some
cases, the native transcriptional regulatory sequence remains on
the vector but is non-functional with respect to transcription of
the gene essential for replication.
[0116] Various combinations of transcriptional regulatory sequences
can be included in a vector. One or more may be heterologous.
Further, one or more may have the tissue-specificity. For example,
a single transcriptional regulatory sequence could be used to drive
replication by more than one gene essential for replication. This
is the case, for example, when the gene product of one of the genes
drives transcription of the further gene(s). An example is a
heterologous promoter linked to a cassette containing an E1a coding
sequence (E1a promoter deleted) and the entire E1b gene. In such a
cascade, only one heterologous transcriptional regulatory sequence
may be necessary. Thus, a single transcriptional regulatory
sequence may drive transcription of the further gene(s) as one
mRNA, as the genes are linked by an internal ribosome entry site
(IRES). When genes are individually (separately) controlled,
however, more than one transcriptional regulatory sequence can be
used if more than one such gene is desired to control
replication.
[0117] The vectors of the present invention, therefore, also
include transcriptional regulatory sequence combinations wherein
there is more than one heterologous transcriptional regulatory
sequence, but wherein one or more of these is not tissue-specific.
For example, one transcriptional regulatory sequence can be a basal
level constitutive transcriptional regulatory sequence. For
example, a tissue-specific enhancer can be combined with a basal
level constitutive promoter.
[0118] The term "tissue-specific gene regulatory region" or
"tissue-specific regulatory region" or "tissue-specific promoter"
or "tissue-specific promoter/enhancer" refers to transcription
and/or translation regulatory regions that function selectively or
preferentially in a specific cell type. Selective or preferential
function confers specificity to the gene therapy treatment since
the therapeutic gene will be primarily expressed in a targeted or
specific cell type. Specific regulatory regions include
transcriptional, mRNA maturation signals and translational
regulatory regions that are cell type specific. Transcriptional
regulatory regions for tumors include, for example, promoters,
enhancers, silencers, or artificial control elements added to the
vector. Examples are steroid hormone receptor and/or response
elements controlled by steriod hormones. Specific examples of such
transcriptional regulatory regions for tumors include the
promoter/enhancer elements for alpha-fetoprotein, carcinoembryonic
antigen and prostate specific antigen. RNA processing signals
include, for example, tissue-specific intron splicing signals,
whereas translational and regulatory signals can include, for
example, mRNA stability signals and translation initiation signals.
Thus, specific regulatory regions include all elements that are
essential for the production of a mature gene product in a specific
cell type.
[0119] The invention is particularly directed to neoplastic cells.
The neoplastic phenotype is characterized by altered morphology,
faster growth rates, higher saturation density, growth in soft agar
and tumorigenicity. The therapeutic genes described herein encode
proteins which exhibit this activity. "Tumorigenicity" is intended
to mean having the ability to form tumors or capable of causing
tumor formation and is synonymous with neoplastic growth.
"Malignancy" is intended to describe a tumorigenic cell having the
ability to metastasize and endanger the life of the host organism.
"Hyperproliferative phenotype" is intended to describe a cell
growing and dividing at a rate beyond the normal limitations of
growth for that cell type. "Neoplastic" also is intended to include
cells lacking endogenous functional tumor suppressor protein or the
inability of the cell to express endogenous nucleic acid encoding a
functional tumor suppressor protein.
[0120] The invention can provide tumor-specific replication
competent vectors wherein the gene regulatory regions include, but
are not limited to alpha-fetoprotein promoter/enhancer, the
carcinoembryonic antigen promoter/enhancer, the tyrosinase
promoter/enhancer and the prostate-specific antigen
promoter/enhancer. It is to be understood that any regulatory or
tumor-specific sequence can be used. For other diseases such as
inflammatory conditions, the inducer could be TNF-.alpha. and the
responding regulatory element the interleukin-6 (IL-6) promoter.
The therapeutic gene can encode interleukin- 10 (IL-10) or another
anti-inflammatory cytokine.
[0121] The vectors useful in the methods of this invention can
replicate specifically in specific tumor cells. The tumor
specificity results from the incorporation of tumor-specific gene
regulatory regions which drive the expression of one or more genes
which are essential for replication. Such elements include, for
example, the alpha-fetoprotein promoter/enhancer, the
carcinoembryonic antigen promoter/enhancer, the tyrosine
promoter/enhancer and the prostate-specific antigen
promoter/enhancer. Each of these gene regulatory regions functions
preferentially in specific tumor cell types. For example, the
alpha-fetoprotein promoter/enhancer functions preferentially in
hepatocellular carcinoma tumor cells. The carcinoembryonic antigen
promoter/enhancer functions preferentially in colon cancer and
breast tumor cells. The prostate-specific antigen promoter/enhancer
functions in prostate tumor cells. The tyrosine promoter enhancer
preferentially functions in melanoma tumor cells. Thus, the
invention provides for the treatment of cancers including, for
example, breast cancer, colorectal cancer, hepatocellular carcinoma
and melanoma cancer.
Vectors
[0122] The present invention is generally directed to an expression
vector capable of expressing one or more heterologous genes in a
modulated and tissue-specific manner wherein a first coding
sequence, derived from a gene that is essential for vector
replication is operably linked to a tissue-specific transcriptional
regulatory sequence, and wherein the vector contains one or more
additional heterologous coding sequences.
[0123] The first coding sequence for a gene product essential for
replication and the tissue-specific transcriptional regulatory
sequence are not derived from the same gene (i.e., are heterologous
to one another). The tissue-specific transcriptional regulatory
sequence, in other words, is not derived from native vector
sequences. For example, when the vector is an adenovirus vector,
the tissue-specific transcriptional regulatory sequence is not
derived from adenovirus.
[0124] The additional heterologous coding sequences can be
expressed from various locations on the vector. For example, the
coding sequence could be linked to the first coding sequence so
that expression is directly dependent on transcription from that
regulatory sequence through the first coding sequence and then into
the additional coding sequence. Alternatively, the additional
coding sequence could be expressed by being operably linked to a
separate transcriptional regulatory sequence that is activated by
the gene product of the first coding sequence (as in
transactivation, for example). As a further alternative, the
additional coding sequence could be placed under the control of a
separate copy of the tissue-specific transcriptional regulatory
sequence to which the first coding sequence is linked, although not
located in proximity to the first coding sequence. Finally, the
additional coding sequence could be, in some instances, transcribed
from its native transcriptional regulatory sequence (e.g.,
promoter) or other different transcriptional regulatory sequence,
different from the one to which the first coding sequence is
operably linked and not activated by the gene product from the
first coding sequence.
[0125] When there are multiple heterologous genes expressed, the
vector could contain various permutations of the above arrangement.
For example, in the case where there is only one additional gene,
this gene could be under the control of the same transcriptional
regulatory sequence controlling the first coding sequence, under
control of a transactivatable transcriptional regulatory sequence,
or transcriptionally linked to the first coding sequence so that
they are transcribed as a unit from the tissue-specific
transcriptional regulatory sequence controlling the first coding
sequence, or, finally, under control of another transcriptional
regulatory sequence, for example its own native transcriptional
regulatory sequence or another constitutive transcriptional
regulatory sequence. When a second additional heterologous gene is
expressed from the vector, the permutations increase but follow the
same general strategy. For example, all three coding sequences can
be transcribed from the same unit linked to a single
tissue-specific transcriptional regulatory sequence. Alternatively,
two of these may be transcribed from the unit and a third
transcribed from the same transcriptional regulatory sequence but
located in a different proximity. Alternatively, the first coding
sequence may be transcribed separately from the second two
sequences but both transcribed from the same tissue-specific
transcriptional regulatory sequence, etc. Alternatively, the first
coding sequence can be used to transactivate the second and third
genes, which can be linked under the control of onetranscriptional
regulatory sequence or under separate transactivatible
transcriptional regulatory sequences. FIG. 8 illustrates some of
the possible permutations that are encompassed in the present
invention. The person of ordinary skill in the art would appreciate
the possible permutations even if not explicitly described
herein.
[0126] In a preferred embodiment, multiple genes on the vector can
be controlled through one transactivator. Accordingly, where the
first coding sequence has transactivator function, activation at
the tissue-specific transcriptional regulatory sequence results in
expression in that function and thus transactivation of any genes
placed under control of transactivatable transcriptional regulatory
sequences at one or more sites in the vector. For example, in an
adenoviral-based vector, by linking a heterologous regulatory
element to the E1a gene in an otherwise replication competent
adenoviral vector, the invention provides the ability to place
genes in other transcriptional regulatory sequences, such as E3,
E4, E1 regions transactivated by E1a, or multiple copies of these
promoters, such as two or more E3 promoters. The design will depend
upon the intended use. For example, for cancer therapy a
tumor-specific transcriptional regulatory sequence would be
desirable to drive E1a expression while therapeutic genes could be
placed under transcriptional regulatory sequences transactivated by
E1a , such as one or more E3 promoters, one or more E4 promoters,
E1 promoters, or combinations thereof, that contain genes such as
HSV-TK, GM-CSF, and IL-2.
[0127] In highly preferred embodiments, vectors are designed to
express a heterologous gene that is toxic for viral replication.
Through this gene one is able to modulate the amount of replication
and thus expression of other heterologous gene products,
therapeutic or otherwise, for maximal benefit and specificity. For
example, in adenoviral vectors having an E1a coding sequence linked
to a tissue-specific promoter and having the HSV-TK gene under
control of the E3 promoter, replication could be controlled by the
addition of ganciclovir.
[0128] The preferred vectors of the present invention are
adenoviral vectors. In a preferred embodiment of the invention, an
adenovirus vector contains a tissue-specific transcriptional
regulatory sequence linked to a gene in the E1 region.
[0129] In one embodiment, both E1a and E1b are operably linked to
heterologous tissue-specific transcriptional regulatory sequences.
In an alternative embodiment, only E1a is linked; E1b remains
intact. In still another embodiment, E1b is linked, and E1a remains
intact or is deleted. In any case, one or more tissue-specific and
promoter-specific cellular transcriptional regulatory factors
allows virus replication to proceed by transcribing the E1a and/or
E1b gene functionally linked to the promoter. Further, either one
or both of the E1b functions may be linked to the transcriptional
regulatory sequence.
[0130] In alternative embodiments, adenovirus vectors are provided
with any of the other genes essential for replication, such as E2
and E4, under control of a heterologous transcriptional regulatory
sequence.
[0131] The invention further embodies the use of plasmids and
vectors having only the essential regions of adenovirus needed for
replication with either E1a , E1b 19 kDa gene, or E1b 55 kDa gene,
or some combination thereof, modified. Such a plasmid, lacking any
structural genes, would be able to undergo DNA replication.
Accordingly, the vectors of the invention may consist essentially
of the transcriptional regulatory sequence and one or more genes
essential for replication of the vector. In the case of viral
vectors, the vectors may consist essentially of the transcriptional
regulatory sequence and the gene or genes essential for replication
or life-cycle functions of the virus. It is also understood that
these vectors may also further consist essentially of a DNA
sequence encoding one or more toxic heterologous gene products when
such vectors are intended as expression vectors for treatment.
[0132] In broader embodiments, the vector is derived from another
DNA tumor virus. Such viruses generally include, but are not
limited to, Herpesviruses (such as Epstein-Barr virus,
cytomegalovirus, Herpes zoster, and Herpes simplex),
papillomaviruses, papovaviruses (such as polyoma and SV40), and
hepatitis viruses, parvoviruses, and picornaviruses.
[0133] The alternative viruses preferably are selected from any
group of viruses in which the essential genes for replication of
the virus can be placed under the control of a tissue-specific
transcriptional regulatory sequence. All serotypes are included.
The only common property of such viruses, therefore, is that they
are transducible into target tissue, are genetically manipulatable,
and are non-toxic when not replicating.
[0134] The relevant viral gene(s) are those that are essential for
replication of the viral vector or of the virus. Examples of genes
include, but are not limited to, the E6 and E7 regions of human
papilloma virus, 16 and 18, T antigen of SV40, and CMV immediate
early genes, polymerases from retroviruses and the like.
Essentially, these include any gene that is necessary for the life
cycle of the virus.
[0135] In further embodiments, the vector is derived from an RNA
virus. In still further embodiments, the vector is derived from a
retrovirus. It is understood, however, that potentially any
replicating vector can be made and used according to the essential
design disclosed herein.
[0136] The vectors described herein can be constructed using
standard molecular biological techniques. Standard techniques for
the construction of such vectors are well-known to those of
ordinary skill in the art, and can be found in references such as
Sambrook et al., in Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor, N.Y. (1989), or any of the myriad of laboratory
manuals on recombinant DNA technology that are widely available. A
variety of strategies are available for ligating fragments of DNA,
the choice of which depends on the nature of the termini of the DNA
fragments and can be readily determined by the skilled artisan.
[0137] An adenovirus vector, in a preferred embodiment, is
constructed first by constructing, according to standard
techniques, a shuttle plasmid which contains, beginning at the 5'
end, the "critical left end elements," which include an adenoviral
5' ITR, an adenoviral encapsidation signal, and an E1a enhancer
sequence; a promoter (which may be an adenoviral promoter or a
foreign promoter); a tripartite leader sequence, a multiple cloning
site (which may be as herein described); a poly A signal; and a DNA
segment which corresponds to a segment of the adenoviral genome.
Such DNA segment serves as a substrate for homologous recombination
with a modified or mutated adenovirus. The plasmid may also include
a selectable marker and an origin of replication. The origin of
replication may be a bacterial origin of replication.
Representative examples of such shuttle plasmids include pAVS6, as
discussed herein and see Trapnell, B. et al., Adv. Drug Deliv. Rev
12: 185-189 (1994). A desired DNA sequence containing a
heterologous gene may then be inserted into the multiple cloning
site to produce a plasmid vector.
[0138] This construct then is used to produce an adenoviral vector.
Homologous recombination then is effected with a modified or
mutated adenovirus in which one or more of the native
transcriptional regulatory sequences have been deleted and replaced
with the desired transcriptional regulatory sequence. Such
homologous recombination may be effected through co-transfection of
the plasmid vector and the modified adenovirus into a helper cell
line by CaPO.sub.4 precipitation.
[0139] Through such homologous recombination, a vector is formed
which includes adenoviral DNA free of one or more of the native
transcriptional regulatory sequences. This vector may then be
transfected into a helper cell line for viral replication and to
generate infectious viral particles. Transfections may take place
by electroporation, calcium phosphate precipitation,
microinjection, or through proteoliposomes.
[0140] The vector may include a multiple cloning site to facilitate
the insertion of DNA sequence(s) containing the heterologous gene
into the cloning vector. In general, the multiple cloning site
includes "rare" restriction enzyme sites; i.e., sites which are
found in eukaryotic genes at a frequency of from about one in every
10,000 to about one in every 100,000 base pairs. An appropriate
vector is thus formed by cutting the cloning vector by standard
techniques at appropriate restriction sites in the multiple cloning
site, and then ligating the DNA sequence containing the
heterologous gene into the cloning vector.
[0141] The coding sequence whose gene product is essential for
vector replication is under the control of a suitable
tissue-specific transcriptional regulatory sequence, which may be a
promoter or an enhancer. A tissue-specific promoter may be, but is
not limited to, AFP, PSA, CEA, DE3, .alpha.-fetoprotein, Erb-B2,
surfactant, and the tyrosinase promoter. A tissue-specific enhancer
may be, but is not limited to, DF3 breast cancer-specific enhancer,
enhancers from viruses, and the steroid receptor family.
[0142] Suitable promoters for expressing the DNA sequence encoding
the heterologous gene product include, but are not limited to,
viral promoters, such as the adenoviral major late promoter,
cytomegalovirus (CMV) promoter, Rous sarcoma virus promoter,
inducible promoters, such as the MMTV promoter, the metallothionein
promoter, heat shock promoters, the albumin promoter, the ApoE
promoter, and the ApoAI promoter. It is to be understood, however,
the scope of the present invention is not limited to specific
heterologous genes or promoters.
[0143] Suitable native promoters (already located on the viral
vector) include, but are not limited to, adenovirus E2, E3 and E4.
Further, as discussed, the tissue-specific transcriptional
regulatory sequence used to activate vector replication can also be
used to control transcription of one or more heterologous genes on
locations separate from the first coding sequence in the vector
genome.
[0144] In one embodiment, the adenovirus may be constructed by
using a yeast artificial chromosome (or YAC) containing an
adenoviral genome according to the method described in Ketner, et
al., Proc. Nat. Acad. Sci. 91:6186-6190 (1994), in conjunction with
the teachings contained herein. In this embodiment, the adenovirus
yeast artificial chromosome is produced by homologous recombination
in vivo between adenoviral DNA and yeast artificial chromosome
plasmid vectors carrying segments of the adenoviral left and right
genomic termini. A DNA sequence containing the heterologous gene
then may be cloned into the adenoviral DNA. The modified adenoviral
genome then is excised from the adenovirus yeast artificial
chromosome in order to be used to generate infectious adenoviral
particles.
[0145] The infectious viral particles may then be administered in
vivo to a host. The host may be an animal host, including
mammalian, non-human primate, and human hosts.
[0146] The viral particles may be administered in combination with
a pharmaceutically acceptable carrier suitable for administration
to a patient. The carrier may be a liquid carrier (for example, a
saline solution), or a solid carrier, such as, for example,
microcarrier beads.
Modulation
[0147] For the methods and particularly treatment methods described
herein, modulating the amount of vector replication is a highly
preferred aspect of the invention. This provides controlled amounts
of a heterologous gene product.
[0148] The vectors herein provide a method for amplifying the
expression of the heterologous gene. In addition to providing
constitutive expression of a heterologous gene (as from a native
promoter or other transcriptional regulatory sequence or from a
tissue-specific promoter in the target tissue), via replication,
the vector provides further copies of the heterologous gene for
amplified expression. Including a toxic gene on the vector provides
a way to control or modulate amplification and thus,
expression.
[0149] Expression of a gene, particularly a toxic gene, can be
absolutely controlled by conditioning the transcription of the gene
completely on replication. This is the case when expression of such
a gene depends upon transactivation from a gene product produced
from a coding sequence of a gene essential for replication operably
linked to a tissue-specific transcriptional regulatory
sequence.
[0150] Vectors expressing gene products, such as HSV-TK, allow the
control of replication by drugs such as ganciclovir. This
expression in conjunction with tissue-specific control of the
expression of a gene essential for replication allows the control
of replication by the administration of an external drug. For
example, tumor cells can be transduced with a vector expressing E1a
in a tissue-specific manner, which vector contains the HSV-TK gene.
Upon replication, HSV-TK is either expressed or expression is
amplified by replication of the vector. If in the course of
monitoring vector replication, there is undesirable spread beyond
the tumor edges into surrounding normal tissues, or release into
the bloodstream which can be monitored, then viral replication can
be dampened by adding ganciclovir to protect the normal cells. At
high doses of ganciclovir (for example, 10-50 .mu.M), replication
can be eliminated completely. At lower doses, replication can be
dampened. In addition, since E1a expression is dependent upon
tissue-specific transcriptional regulatory sequences, in normal
cells, E1a expression would be prevented. Therefore, TK expression
would also be diminished. Further, in those embodiments in which
the TK coding sequence is operably linked to either the
tissue-specific promoter or a promoter dependent on E1a
transactivation, TK expression should cease entirely.
[0151] Also, since most normal cells are not actively dividing, the
addition of ganciclovir should shut down viral replication but
should not harm cells which would not incorporate this analogue
into cellular DNA. Further, even if the cell were dividing, the
elimination of TK expression would prevent the phosphorylation of
ganciclovir and thus, no deleterious on the cell should result.
[0152] In some embodiments, the gene such as TK does not need to be
expressed from the vector itself, but can be expressed from an
unlinked location such as a separate vector or a cellular
genome.
[0153] It should be appreciated that the ability to modulate viral
replication allows the temporary expression of a given gene, and
particularly a cytotoxic gene. This is useful, for example, in a
treatment context where temporary expression of a gene is necessary
to provide gene therapy. A gene can be expressed at a certain
level, maintained at that level, and then expression can be
increased and the cell eliminated.
[0154] In addition, it may not be desirable to eliminate a cell,
but may be desirable to simply express a gene. In this case, it is
desirable to express a gene at certain levels. Control of gene
expression thus could be effected by a combination of, for example,
E1a , TK, and GCV.
Replication and Expression
[0155] Expression of heterologous genes and/or treatment thereby
are possible using the vectors described herein. Therefore, the
invention generally encompasses methods of replicating the vectors
described herein.
[0156] In preferred embodiments, the methods are specifically
directed to the introduction into a target tissue of a
replication-conditional adenoviral vector. This vector selectively
replicates in the cells of the target tissue. The replication is
conditioned upon the function of a transcriptional regulatory
sequence to which a viral gene is operably linked, which gene is
necessary for vector replication. Thus, in the target tissue,
replication can occur because either a cellular function in the
target tissue allows transcription. Alternatively, there is a
deficiency in a cellular function in the target tissue that
normally prevents or inhibits transcription. The presence or
absence of such functions provides the selectivity that allows the
treatment of a specific tissue with minimum effect on the
surrounding tissue(s).
[0157] The present invention thus provides methods for selectively
distributing a polynucleotide in a given tissue in vivo,
significantly reducing or avoiding distribution in non-target
tissue. The polynucleotide is provided in the
replication-conditional vector which is selectively distributed in
the given tissue.
[0158] The present invention also provides methods for selectively
expressing a gene product in a given tissue, avoiding or
significantly reducing expression in non-target or non-tumor
tissue. In preferred embodiments, the gene expressed from the
vector is a gene that has the potential of being cytotoxic to the
host cell and/or toxic to viral replication in the cell. Thus,
there is the option of obliterating the cell in which the virus is
replicating, or simply decreasing or eliminating viral replication
and avoiding cell killing. Accordingly, the invention provides a
method for expressing such genes, for example thymidine kinase, in
a cell. This provides the possibility of treatment of diseases in
which tissue-specific viral replication and gene expression is
desired. It also thus provides a method for killing a cell in the
case of conditions in which cell killing is desirable, such as
cancer and other diseases involving abnormal cellular proliferation
such as restenosis.
[0159] The invention provides methods for distribution of the
above-mentioned vectors to a greater number of target cells than
would be reached using a non-replicating vector. Successive
infection provides a "domino effect" so that all or substantially
all of the cells in the target tissue are reached. Cells in
addition to those first exposed to the polynucleotide, vector, or
gene product, are thus potentially reached by the methods.
[0160] Such treatment is particularly necessary in cases in which
surgical intervention is not feasible. For example, in patients
with abnormal tissue intimately associated with neural tissue,
surgery may be precluded or highly dangerous. Further, in the case
of multiple metastases or microscopic metastases, surgery is not
feasible.
[0161] In the target tissue, DNA replication alone may occur. Late
viral functions that result in packaging of vector DNA into virions
may also occur.
[0162] The vector may be introduced into the target tissue as naked
DNA or by means of encapsidation (as an infectious virus particle
or virion). In the latter case, the distribution is accomplished by
successive infections of cells in the tissue by the virus such that
substantially all or a significant number of the daughter cells are
infected.
[0163] Tissue specificity is particularly relevant with respect to
targeting an abnormal counterpart of a particular tissue type while
avoiding the normal counterpart of the tissue, or avoiding
surrounding tissue of a different type than the abnormal tissue,
while treating the abnormal tissue. For example, the vectors of the
present invention are useful for treating metastases to the liver.
One specific example is colon cancer, which often metastasizes into
the liver. It has been found that even when colon cancer
metastasizes into the liver, the CEA promoter is active in the
cells of the metastases but not in normal liver cells. Accordingly,
normal human adult liver should not support replication of a virus
that has viral genes essential for replication linked to the colon
cancer CEA-specific promoter. Replication should occur in the
primary cancer cells. Another example is breast cancer, which also
metastasizes to the liver. In this case, the DF3 mucin enhancer is
linked to a gene essential for replication such as both E1a and
E2a. Replication should occur in breast cancer but not in normal
liver. A further example is the a-fetoprotein promoter, which is
active in hepatocellular carcinoma. This promoter is linked to a
gene essential for replication. It has been found that the promoter
is active only in the hepatocellular carcinoma. Accordingly, a
virus is used that has a gene essential for replication linked to
this promoter. Replication should be limited to hepatocellular
carcinoma. A further example is the tyrosinase promoter. This
promoter is linked to a gene essential for replication. Replication
should occur in melanoma and not in normal skin. In each case,
replication is expected in the abnormal but not the normal
cells.
[0164] In a further embodiment of the invention, the vector encodes
a heterologous gene product which is expressed from the vector in
the tissue cells. The heterologous gene product can be toxic for
the cells in the targeted tissue or confer another desired
property.
[0165] A gene product produced by the vector can be distributed
throughout the tissue, because the vector itself is distributed
throughout the tissue. Alternatively, although the expression of
the gene product may be localized, its effect may be more
far-reaching because of a bystander effect or the production of
molecules which have long-range effects such as chemokines. The
gene product can be RNA, such as antisense RNA or ribozyme, or
protein. Examples of toxic products include, but are not limited
to, thymidine kinase in conjunction with ganciclovir.
[0166] A wide range of toxic effects is possible. Toxic effects can
be direct or indirect. Indirect effects may result from the
conversion of a prodrug into a directly toxic drug. For example,
Herpes simplex virus thymidine kinase phosphorylates ganciclovir to
produce the nucleotide toxin ganciclovir phosphate. This compound
functions as a chain terminator and DNA polymerase inhibitor,
prevents DNA synthesis, and thus is cytotoxic. Another example is
the use of cytosine deaminase to convert 5'-fluorocytosine to the
anti-cancer drug 5'-fluorouracil. For a discussion of such
"suicide" genes, see Blaese, R. M. et al., Eur. J. Cancer
30A:1190-1193 (1994).
[0167] Direct toxins include, but are not limited to, diphtheria
toxin (Brietman et al., Mol. Cell Biol. 10:474-479 (1990)),
pseudomonas toxin, cytokines (Blankenstein, T., et al., J. Exp.
Med. 173:1047-1052 (1991), Colombo, M. P., et al., J. Exp. Med.
173:889-897 (1991), Leone, A., et al., Cell 65:25-35 (1991)),
antisense RNAs and ribozymes (Zaia, J. A. et al., Ann. N. Y. Acad.
Sci. 660:95-106 (1992)), tumor vaccination genes, and DNA encoding
for ribozymes.
[0168] In accordance with the present invention, the agent which is
capable of providing for the inhibition, prevention, or destruction
of the growth of the target tissue or tumor cells upon expression
of such agent can be a negative selective marker; i.e., a material
which in combination with a chemotherapeutic or interaction agent
inhibits, prevents or destroys the growth of the target cells.
[0169] Thus, upon introduction to the cells of the negative
selective marker, an interaction agent is administered to the host.
The interaction agent interacts with the negative selective marker
to prevent, inhibit, or destroy the growth of the target cells.
[0170] Negative selective markers which may be used include, but
are not limited to, thymidine kinase and cytosine deaminase. In one
embodiment, the negative selective marker is a viral thymidine
kinase selected from the group consisting of Herpes simplex virus
thymidine kinase, cytomegalovirus thymidine kinase, and
varicella-zoster virus thymidine kinase. When viral thymidine
kinases are employed, the interaction or chemotherapeutic agent
preferably is a nucleoside analogue, for example, one selected from
the group consisting of ganciclovir, acyclovir, and
1-2-deoxy-2-fluoro-D-arabinofuranosil-5-iodouracil (FIAU). Such
interaction agents are utilized efficiently by the viral thymidine
kinases as substrates, and such interaction agents thus are
incorporated lethally into the DNA of the tumor cells expressing
the viral thymidine kinases, thereby resulting in the death of the
target cells.
[0171] When cytosine deaminase is the negative selective marker, a
preferred interaction agent is 5-fluorocytosine. Cytosine deaminase
converts 5-fluorocytosine to 5-fluorouracil, which is highly
cytotoxic. Thus, the target cells which express the cytosine
deaminase gene convert the 5-fluorocytosine to 5-fluorouracil and
are killed.
[0172] The interaction agent is administered in an amount effective
to inhibit, prevent, or destroy the growth of the target cells. For
example, the interaction agent is administered in an amount based
on body weight and on overall toxicity to a patient. The
interaction agent preferably is administered systemically, such as,
for example, by intravenous administration, by parenteral
administration, by intraperitoneal administration, or by
intramuscular administration.
[0173] When the vectors of the present invention induce a negative
selective marker and are administered to a tissue or tumor in vivo,
a "bystander effect" may result, i.e., cells which were not
originally transduced with the nucleic acid sequence encoding the
negative selective marker may be killed upon administration of the
interaction agent. Although the scope of the present invention is
not intended to be limited by any theoretical reasoning, the
transduced cells may be producing a diffusible form of the negative
selective marker that either acts extracellularly upon the
interaction agent, or is taken up by adjacent, non-target cells,
which then become susceptible to the action of the interaction
agent. It also is possible that one or both of the negative
selective marker and the interaction agent are communicated between
target cells.
[0174] In one embodiment, the agent which provides for the
inhibition, prevention, or destruction of the growth of the tumor
cells is a cytokine. In one embodiment, the cytokine is an
interleukin. Other cytokines which may be employed include
interferons and colony-stimulating factors, such as GM-CSF.
Interleukins include, but are not limited to, interleukin- 1,
interleukin-1.beta., and interleukins-2-15. In one embodiment, the
interleukin is interleukin-2.
[0175] In a preferred embodiment of the invention, the target
tissue is abnormally proliferating, and preferably tumor tissue.
The vector or virus is distributed throughout the tissue or tumor
mass.
[0176] All tumors are potentially amenable to treatment with the
methods of the invention. Tumor types include, but are not limited
to hematopoietic, pancreatic, neurologic, hepatic, gastrointestinal
tract, endocrine, biliary tract, sino-pulmonary, head and neck,
soft tissue sarcoma and carcinoma, dermatologic, reproductive
tract, and the like. Preferred tumors for treatment are those with
a high mitotic index relative to normal tissue. Preferred tumors
are solid tumors, and especially, tumors of the brain, most
preferably glioma.
[0177] The methods can also be used to target other abnormal cells,
for example, any cells which are harmful or otherwise unwanted in
vivo. Broad examples include cells causing autoimmune disease,
restenosis, and scar tissue formation, abnormal angiogenesis, PRD,
arthritis, chronic diabetes, and ARMD.
[0178] Further, treatment can be ex vivo. Ex vivo transduction of
tumor cells would overcome many of the problems with current viral
delivery systems. Tissue is harvested under sterile conditions,
dissociated mechanically and/or enzymatically and cultured under
sterile conditions in appropriate media. Vector preparations
demonstrated to be free of endotoxins and bacterial contamination
are used to transduce cells under sterile conditions in vitro using
standard protocols. The accessibility of virus to cells in culture
is currently superior to in vivo injection and permits introduction
of vector viral sequences into essentially all cells. Following
removal of virus-containing media cells are immediately returned to
the patient or are maintained for several days in culture while
testing for function or sterility is performed.
[0179] For example, patients with hypercholesterolemia have been
treated successfully by removing portions of the liver, explanting
the hepatocytes in culture, genetically modifying them by exposure
to retrovirus, and re-infusing the corrected cells into the liver
(Grossman et al., Nature Genetics 6:335-341 (1994)).
[0180] Viral transduction also has potential applications in the
area of experimental medicine. Transient expression of biological
modifiers of immune system function such as IL-2, IFN-.gamma.,
GM-CSF or the B7 co-stimulatory protein has been proposed as a
potential means of inducing anti-tumor responses in cancer
patients.
[0181] In broader embodiments, the vector is derived from another
DNA tumor virus. Such viruses generally include, but are not
limited to, Herpesviruses (such as Epstein-Barr virus,
cytomegalovirus, Herpes zoster, and Herpes simplex),
papillomaviruses, papovaviruses (such as polyoma and SV40), and
hepatitis viruses.
[0182] The relevant viral gene(s) are those that are essential for
replication of the viral vector or of the virus. Examples of genes
include, but are not limited to, the E6 and E7 regions of human
papilloma virus, 16 and 18, T antigen of SV40, and CMV immediate
early genes, polymerases from retroviruses and the like.
Essentially, these include any gene that is necessary for the life
cycle of the virus.
[0183] In further embodiments, the vector is derived from an RNA
virus. In still further embodiments, the vector is derived from a
retrovirus. It is understood, however, that potentially any
replicating vector can be made and used according to the essential
design disclosed herein.
Diagnostic
[0184] It is important to know whether the vectors of the invention
will replicate in a specific tissue from a patient. If vector
replication is found to be beneficial for therapy, then a screen is
provided for those patients who best respond to the therapy
disclosed herein. If it is found to be harmful, then there is a
screen for prevention of the treatment of patients who would have
an adverse response to the treatment. Currently, the only
non-biological assays that are commonly used are expression
screening, PCR, and sequencing. These often result in false
negatives, are time-consuming, expensive, and yield only
information in the best of cases about the status of the genes and
not their biological function.
[0185] Accordingly, a method is provided for identifying an
abnormal tissue, the cells of which contain a transcription factor
that allows replication of a replication-conditional vector, or are
deficient for an inhibitory factor for transcription.
[0186] In this method, a tissue biopsy is explanted, a
replication-conditional vector is introduced into the cells of the
biopsy, and vector DNA replication in the cells is quantitated.
Accordingly, a method is provided for screening tissue for the
presence of factors that allow vector replication, or for a
deficiency of a factor that inhibits transcription. Such a screen
is useful, among other things, for identifying tissue, prior to
treatment, which will be amenable to treatment with a particular
vector to be replicated in the tissue.
[0187] Therefore, a method is provided for assaying vector utility
for treatment by removing a tissue biopsy from a patient,
explanting the biopsy into tissue culture, introducing the
replication-conditional vector into the biopsy, and assaying vector
replication in the cells of the biopsy.
[0188] Testing or screening of tissues includes an assay for vector
nucleic acid replication or for virus replication, when the vector
is capable of forming infectious virions.
[0189] Thus, the invention provides a method for screening a tumor
for transcription regulatory functions that allow vector
replication or for the absence of these functions which would
normally prevent the replication of a virus vector.
[0190] However, any abnormal tissue can be screened for the
functions described above by an assay for nucleic acid or virus
replication.
Producer Cells
[0191] In a further embodiment of the invention, a cell is provided
which contains a virion produced in the cell by replication in the
cell of the replication-conditional vectors of the present
invention. Thus, the invention provides "producer cells" for the
efficient and safe production of recombinant
replication-conditional vectors for further use for targeted gene
therapy in vivo.
[0192] One of the major problems with the currently available
producer cells is that such cells contain, in the genome, viral
sequences that provide complementing functions for the replicating
vector. Because the cell contains such sequences, homologous
recombination can occur between the viral sequence in the genome
and the viral vector sequences. Such recombination can regenerate
recombinant wild-type viruses which contaminate the vector or virus
preparation produced in the producer cell. Such contamination is
undesirable, as the wild-type viruses or vectors can then replicate
in non-target tissue and thereby impair or kill non-target cells.
Therefore, one of the primary advantages of the producer cells of
the present invention is that they do not contain endogenous viral
sequences homologous to sequences found in the vector to be
replicated in the cells. The absence of such sequences avoids
homologous recombination and the production of wild-type viral
recombinants that can affect non-target tissue.
[0193] Accordingly, the invention embodies methods for constructing
and producing replication-conditional virions in a cell comprising
introducing the replication-conditional vector of the present
invention into the cell wherein the genome of the cell is devoid of
vector sequences, replicating the vector in the cell, forming the
virion, and purifying the virion from the cell. Preferred vectors
are DNA viral vectors, including but not limited to herpesvirus,
papillomavirus, hepatitis virus, and papovavirus vectors. In
preferred embodiments of the invention, the virion is an adenoviral
virion and the vector is an adenoviral vector. In further
embodiments of the invention, the cell is a tumor cell.
[0194] In a further preferred embodiment, the vector encodes a
heterologous gene product such that the virion also encodes the
gene product, and when the vector or virion are used for gene
therapy, the therapy is facilitated by expression of the
heterologous gene product. Alternatively, the producer cell can be
used for the production of a heterologous gene product per se
encoded by the vector. When the vector replicates in the producer
cell, the gene product is expressed from the multiple copies of the
gene encoding the gene product. Following expression, the gene
product can be purified from the producer cells by conventional
lysis procedures, or secreted from the producer cell by appropriate
secretion signals linked to the heterologous gene by known methods.
The transduction of cells by adenoviral vectors has been described.
Transfection of plasmid DNA into cells by calcium phosphate
(Hanahan, D., J. Mol. Biol. 166:577 (1983)), lipofection (Feigner
et al., PNAS 84:7413 (1987)), or electroporation (Seed, B., Nature
329:840 ( )) has been described. DNA, RNA, and virus purification
procedures are described (Graham et al., J. Gen. Virol. 36:59-72
(1977).
[0195] Preferred hosts for producer cell lines include but are not
limited to HuH7, SW480, BIGF10, HepG2, MCF-7, and SK-MEL2. Primary
tumors from which cell lines can be derived, or existing cell
lines, can be tested for the ability to allow replication by means
of the tissue-specific transcriptional regulatory sequence. Any
primary tumor could be explanted and developed into producer cells
for the vectors of the present invention. As long as the cell does
not contain endogenous vector or viral sequences that could
recombine with the vector or virus to produce wild-type vector or
virus, the cell is potentially useful as a host. It is understood
that any cell is potentially useful, not only tumor cells.
[0196] The ultimate goal for a producer cell line, and particularly
an adenoviral producer line, is to produce the highest yield of
vector with the least possibility of contamination by wild-type
vector. Yield depends upon the number of cells infected. Thus, the
more cells that it is possible to grow and infect, the more virus
it is possible to generate. Accordingly, candidate cells would have
a high growth rate and will grow to a high density. The cell should
also have a high amount of viral receptor so that the virus can
easily infect the cell. Another characteristic is the quality of
the vector produced (i.e., the preparation should not include a
high amount of non-infectious viral particles). Accordingly,
candidate producer cells would have a low
particle-to-plaque-forming-unit ratio. Thus, these cells are a
preferred cell type for deriving a producer cell line. Primary
explants or the known cell lines can be used.
[0197] Thus, such obtainable cells can serve as producer cells for
recombinant replication-conditional vectors, viruses, and gene
products.
Introduction of Vectors into Cells
[0198] A variety of ways have been developed to introduce vectors
into cells in culture, and into cells and tissues of an animal or a
human patient. Methods for introducing vectors into mammalian and
other animal cells include calcium phosphate transfection, the
DEAE-dextran technique, microinjection, liposome mediated
techniques, cationic lipid-based techniques, transfection using
polybrene, protoplast fusion techniques, electroporation and
others. These techniques are well known to those of skill, are
described in many readily available publications and have been
extensively reviewed. Some of the techniques are reviewed in
Transcription and Translation, A Practical Approach, Hames, B. D.
and Higgins, S. J., eds., IRL Press, Oxford (1984), herein
incorporated by reference in its entirety, and Molecular Cloning,
Second Edition, Maniatis et al., Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y (1989), herein incorporated by
reference in its entirety.
[0199] Several of these techniques have been used to introduce
vectors into tissues and cells in animals and human patients. Chief
among these have been systemic administration and direct injection
into sites in situ. Depending on the route of administration and
the vector, the techniques have been used to introduce naked DNA,
DNA complexed with cationic lipid, viral vectors and vector
producer cell lines into normal and abnormal cells and tissues,
generally by direct injection into a targeted site.
[0200] The aforementioned techniques for introducing
polynucleotide, viral and other vectors into cells in culture, in
animals and in patients can be used to develop, test and produce,
as well as use vectors in accordance with the invention. For
instance, cells containing a vector introduced by these methods can
be used for producing the vector. In addition, cells containing a
vector can be used as producer-cells and introduced into cells or
tissues of an animal to produce the vector in situ.
Assay of DNA and Viral Replication
[0201] Replication of a polynucleotide, viral or other vector can
be assayed by well-known techniques. Assays for replication of a
vector in a cell generally involve detecting a polynucleotide,
virions or infective virus. A variety of well-known methods that
can be used for this purpose involve determining the amount of a
labeled substrate incorporated into a polynucleotide during a given
period in a cell.
[0202] When replication involves a DNA polynucleotide,
.sup.3H-thymidine often is used as the labelled substrate. In this
case, the amount of replication is determined by separating DNA of
the vector from the bulk of cellular DNA and measuring the amount
of tritium incorporate specifically into vector DNA.
[0203] Other methods to assay replication, however, include, but
are not limited to, hexon immunohistochemistry or another late gene
immunoassay that is linked to DNA or viral replication.
[0204] Replication of a polynucleotide vector also may be detected
by lysing or permeating cells to release the polynucleotide, then
isolating the polynucleotide and quantitating directly the DNA or
RNA that is recovered. Polynucleotide replication also may be
detected by quantitative PCR using primers that are specific for
the assay polynucleotide.
[0205] Virions may be assayed by EM counting techniques well known
to the art, by isolating the virions and determining protein and
nucleic acid content, and by labeling viral genomic polynucleotides
or virion proteins and determining the amount of virion from the
amount of polynucleotide or protein.
[0206] It is well known that virions may not all be viable and
where infectivity is important, infectious titer may be determined
by cytopathic effect or plaque assay.
[0207] Any of these well-known techniques, among others, can be
employed to assay replication of a vector in a cell or tissue in
accordance with the invention. It will be appreciated that
different techniques will be better suited to some vectors than
others and to some cells or tissues than others.
[0208] Having thus described herein the invention in general terms,
the following non-limiting examples are presented to illustrate the
invention. Example 1 shows the replacement of the constitutive E1A
promoter on an adenoviral vector with a tumor-specific promoter.
Constructs made this way have the E1a protein expressed only in
tumor cells and therefore, will replicate only in tumor cells.
EXAMPLE 1
[0209] The Hepatoma-Specific Promoter, .alpha.-Fetoprotein
Promoter, Linked to E1a
[0210] The .alpha.-fetoprotein (AFP) promoter has been previously
shown to be highly active in hepatoma cells and silent in adult
hepatocytes and other adult tissues. A 4.9 kb .alpha.-fetoprotein
promoter-containing construct was used to derive the promoter.
Alternatively, the promoter could also be made based on available
references.
[0211] pAVE1a02i (FIG. 1A) which places the E1a/E1b genes under the
control of the .alpha.-fetoprotein promoter in an adenovirus
shuttle plasmid was cloned by purifying a restriction fragment
which contains the E1a coding region only and all of E1b gene by
cleaving the plasmid 380-280E1 (FIG. 1A) with SpeI and MunI and
ligating this to pAVSAFP.TK1 (FIG. 1A) cleaved with MunI and
NheI.
[0212] The adenovirus shuttle plasmid pAVSAFP.TK1 (FIG. 1A), which
has the TK gene under the control of the native 4.9 kb
.alpha.-fetoprotein promoter, was made exactly as described in
FIGS. 11 and 12 of U.S. patent application Ser. No. 08/444,284,
Chiang et al., "Gene therapy of hepatocellular carcinoma through
cancer-specific gene expression," filed on May 18, 1995, which is
incorporated herein by reference for its relevant teaching. This
shuttle plasmid contains the left ITR, packaging signal, the native
AFP promoter, the HSV TK gene, and a homologous recombination
fragment. Digestion of this plasmid with MunI and NheI removes the
TK gene and part of the E1a gene. What remains is a 10,667 base
pair fragment. This fragment is added to the E1a and E1b open
reading frames from plasmid 380-280E1 (see below).
[0213] Plasmid 380-280E1 contains the E1a ORF and all of E1b. A
SpeI/MunI restriction fragment of 3397 base pairs as described in
FIG. 1A from plasmid 380-280E1 was used to ligate to the MunI/NheI
fragment from pAVSAFP.TK1 to construct pAVE1a02i. The SpeI/MunI
fragment from 380-280E1 can be found by reference to plasmid
SE280-E1 which contains the same fragment as found in 380-280E1;
SE280-E1 can be found in U.S. patent application Ser. No.
08/458,403, incorporated herein by reference for its relevant
teaching.
[0214] The shuttle plasmid pAVE1a04i (FIG. 1C) was cloned by
digesting pAVE1a02i (FIG. 1A) with AscI followed by DNA polymerase
I large fragment (Klenow) filling of the 3' recessed end (FIG. 1A).
The linearized pAVE1a02i was then digested with SpeI (FIG. 1A) and
the resulting 9104 base pair fragment was isolated by agarose gel
electrophoresis. This procedure removes the AFP promoter so that
the 9104 base pair fragment contains the above-mentioned components
but no promoter.
[0215] The plasmid pAF(AB).sub.2(S).sub.6-CAT (FIG. 1A, 1B) was
digested with XbaI followed by Klenow filling of the 3' recessed
ends. pAF(AB).sub.2(S).sub.6-CAT contains a shortened AFP promoter
with six silencer elements and two enhancer regions, AB.sub.2 that
are responsible for enhancing the activity in hepatoma cells and
repressing the activity in adult liver cells.
[0216] pAF(AB).sub.2(S).sub.6-CAT was constructed by placing six
copies of the distal silencer immediately upstream of the basal 200
base pair AFP promoter. Two copies of the enhancer AB region,
originally thought to be in opposite orientation, were placed
immediately upstream of the silencer elements. The distal silencer
element, the basal promoter, and the enhancer elements are as
described in Nakabayashi et al. (Molec. & Cell. Biol.
11:5885-5893 (1991)).
[0217] The linearized pAF(AB).sub.2(S).sub.6-CAT was then digested
with SpeI (FIG. 1A) and the 1986 base pair fragment containing the
shortened AFP promoter was isolated by gel electrophoresis. The
9104 base pair fragment of pAvE1a02i and the 1986 base pair
fragment of pAF(AB).sub.2(S).sub.6-CAT containing the shortened
synthetic AFP promoter, were ligated to make the plasmid pAvE1a04i
(FIG. 1C) which places the adenovirus E1a gene under the control of
the shortened AFP promoter.
[0218] The pAF(AB).sub.2(S).sub.6-CAT was reported to have two
copies of the enhancer AB region in opposite orientation but was
determined by the inventors to have the enhancer region as a tandem
repeat. The plasmid map of pAvE1a04i shows the enhancer (AB) region
of the AFP promoter as an inverted repeat (FIG. 1A). However, the
map was later corrected to show the true orientation of the
enhancer regions. The corrected plasmid map was designated as
pAvE1a06i.
Construction of a Virus with the Hepatoma-Specific AFP Promoter
Operably Linked to the E1a Gene
[0219] The adenovirus AvE1a04i (FIG. 2C) was constructed by
homologous recombination of the shuttle plasmid, pAvE1a06i (see
FIGS. 1C and 2B), with the large (Cla1) fragment of Av1lacZ DNA
(FIG. 2A) in 293 cells. Resulting recombinant virus containing the
AFP promoter with the two direct repeat enhancers was isolated and
initially designated as AvE1a06i (FIG. 2C, top of page). In the
process of plaque purifying the recombinant virus, a single
enhancer deletion mutant was isolated and designated as AvE1a04i
(FIG. 2C, bottom of page).
[0220] AvE1a04i was shown to replicate specifically in cell lines
as described in PCT App. No. US 95/15455 (U.S. Pat. No.
5,998,205).
[0221] The plasmid pAVE1a04i was grown in STBL2 cells and was
purified by standard cesium banding methods prior to use in
transfection. Genomic AV1lacZ4 DNA was isolated from cesium
gradient-purified virus (herein described). The AV1lacZ4 purified
virus was digested with proteinase K and the DNA isolated by
phenol/chloroform extraction. The purified DNA was digested with
Cla1 and the large fragment was isolated by gel electrophoresis and
quantified. 5 .mu.g of the plasmid pAVE1a04i and 2.5 .mu.g of the
large Cla1fragment of AV1lacZ4 were co-transfected into 293 cells
using a calcium phosphate-mediated transfection procedure (Promega,
E1200 kit). The transfection plate was overlayered with a 1%
agarose overlay and incubated until plaques formed. Once plaques
had formed, they were picked and the virus was released into 500
.mu.l of IMEM media by alternate cycles of freezing and thawing
(5.times.). The eluted viral plaques were reamplified on A30 cells
for 48 hours and then the cells were lysed for use in screening by
PCR.
[0222] Primers specific for the short AFP (sAFP) promoter in
plasmid pAVE1a04i were used to identify the putative plaques. FIG.
9A shows that viral plaques contain a sAFP-specific band of the
predicted molecular weight and specific for the sAFP primers. To
confirm that this recombinant virus was not contaminated with
Ad5dl327 (wild type), E1a primers were used. FIG. 9B demonstrates
that no wild type virus was present and that pAVE1a04i plasmid
sequences were present in the recombinant virus. FIG. 9C
demonstrates that little or no AV1lacZ4 was present. The data
indicate the construction of a virus with E1a under control of a
tissue-specific promoter and that the virus is capable of
replication in A30 cells.
[0223] Individual plaques were grown in A30 cells and analyzed by
PCR for the presence of the AFP promoter (FIG. 9A). The arrow
indicates the AFP-specific band generated from PCR. The figure
shows that the band is present in each of the viruses in the
selected plaques (L6, L10, L11, M1 and M2). The control in the
experiment was an A30 cell lysate, expected not to contain the
band. The experiment also included the PCR reaction with the
plasmid pAVE1a04i (the shuttle plasmid from which the virus was
made and which therefore should produce the AFP-specific fragment).
Thus, FIG. 9A confirms the presence of a recombinant virus
containing the AFP promoter. FIGS. 9B and 9C confirm that these
results were not the result of contamination in the individual
plaques. FIG. 9B uses E1a-specific primers to detect the presence
of any contaminating wild-type virus. The arrow shows the band
produced with E1a-specific primers. The figure shows that none of
the recombinant viruses produced the relevant band. FIG. 9C
confirms that there is no AV1.lacZ contamination in the viral
plaques (since the viruses were made using AV1.lacZ DNA). The
figure indicates that only the lane containing AV1.lacZ DNA
produced the band.
Tissue-Specific Viral Replication
[0224] Cytopathic viral lysate of this virus ("AVAFPE1a") was
serially diluted in logs of 10 on A549.30 cells, A549 cells, and
HuH 7 cells. A549.30 cells express the E1a from the glucocorticoid
receptor element (GRE) promoter in the presence of dexamethasone
since this construct is integrated into the genome of this cell
line. Thus, any E1a-deleted virus or any virus not expressing E1a
should be able to replicate in this cell line. This has previously
been shown for E1-deleted vectors (unpublished communication). As
can be seen from FIGS. 10A and 10D, the AVAFPE1a vector replicates
in the infected cells as indicated by characteristic cytopathic
effects and spreading of cell death. The A549 cells do not express
AFP and should not be capable of transactivating the AFP promoter.
In addition, A549 cells do not express E1a. Thus, AVAFPE1a should
not be able to replicate in this cell line. As can be seen from
FIGS. 10B and 10E, both uninfected and infected wells appear
identical with no characteristic cytopathic effects or spreading
observed at all dilutions tested. HuH 7 cells do express AFP,
should transactivate the AFP promoter, and should make E1a with
subsequent replication. As shown in FIGS. 10C and 10F, AVAFPE1a
clearly replicates, as indicated by the cytopathic effects. In
addition, on several wells of infected HuH 7 cells, the replication
began with a single plaque which spread throughout the rest of the
well within one week. All HuH 7 wells showing cytopathic effects
were tested by PCR and demonstrated to be free of wild-type virus
and AV1LacZ4 virus, and to contain an intact AFP promoter. These
data clearly indicate that a virus has been constructed that is
capable of replicating specifically in tumor cells expressing
AFP.
The Breast Cancer-Specific DF3-Mucin Enhancer
[0225] The DF3 breast carcinoma associated antigen (MUC1) is highly
overexpressed in human breast carcinomas. The expression of the
gene is regulated at the transcriptional level. The DNA sequence
between -485-588 is necessary and sufficient for conferring a
greater than 10-fold increase in transcription of the reporter gene
CAT when placed immediately upstream of a basal promoter derived
from the Herpesvirus TK promoter in transient transfection assays
performed in the human breast cancer cell line MCF-7. A specific
transcription factor which binds to this region of DNA has also
been found within cells derived from the breast cancer cell line
MCF-7 but not a non-breast cancer cell line HL-60. The same region
of DNA has been found to promote breast cancer-specific expression
of the TK gene in the context of a retroviral construct or an
adenoviral construct.
[0226] The DF3 enhancer from -598 to -485 (obtained from GenBank)
was synthesized by constructing four oligonucleotides synthesized
in such a way as they would overlap and anneal. The
oligonucleotides are shown in Table 1. Additional restriction sites
were added on both ends for future ease of cloning. One end was
kept blunt to enable cloning into the SmaI site of the vector
pTK-Luc. This vector contains the basal promoter of the Herpesvirus
TK gene which gives low level basal activity in a variety of cells.
It was used as a source of this basal promoter. The other end had
an overlapping BglII site for ease in cloning into the BglII site
of pTK-Luc. 1,000 ng of each oligonucleotide were annealed in 0.017
M Tris, pH 8.0, 0.16 M NaCl in a total volume of 26.5 .mu.l by
heating at 95.degree. C. for two minutes and allowing to cool to
room temperature after several hours. Finally, 1 .mu.l of this
mixture was ligated to 100 ng of previously SmaI/BglII-and glass
milk (BIO 101)- purified vector by standard conditions. Following
transformation into DH5 cells (GIBCO), colonies were screened for
the presence of the insert by standard restriction digests. DNA
derived from this vector is then cleaved with HindIII and blunted
by Klenow. It is then cleaved by AscI. This fragment, which
contains the DF3 enhancer lined to the basal TK promoter, is then
purified by agarose gel electrophoresis and glass milk and ligated
to the plasmid pAVE1a02i, cleaved with Spe I and blunt-ended with
AscI and purified as above. The resultant plasmid has the E1A gene
product under the control of the DF3 enhancer and basal TK promoter
and is in an adenoviral shuttle plasmid. 5 .mu.g of this plasmid,
pAVE1a03i, is cotransfected with 5 .mu.g of the right ClaI fragment
arm, derived from Add1327, into 293 cells. Plaques are screened for
the expected recombinant virus by standard methods.
[0227] A crude virus lysate is used to infect MCF-7 at an MOI of
10. Virus stocks are confirmed to replicate specifically in breast
cancer cells by standard methods. Virus is scaled up in MCF-7 cells
and/or 293 cells as described for scaleup and purification on 293
cells. Virus stocks are tested for replication in vivo by using a
mode mouse model of MCF-7 and, as a negative control, a cervical
cancer (Hela) derived tumor is used. Virus is tested for a
recombinational event in 293 cells which would generate a wild-type
virus by PCR assay of the original E1A promoter which would only be
in a wild-type virus. A variety of other human and rat breast
cancer cell lines and non-related cell lines are also tested. The
TK gene can be inserted into the E3 region and have TK driven
either by the E1A-dependent promoter present there or under the
control of the RSV or CMV promoter.
The Melanoma-Specific Tyrosinase Promoter
[0228] PCR primers and PCR were used to clone a fragment of DNA 800
bp upstream of the tyrosinase gene from mouse genomic DNA using PFU
and the described primers as described by Stratogene. The resultant
PCR fragment was cloned into pCRSCRIPT and then recloned into
pAVE1a02i by digesting the new plasmid with AscI/SpeI and pAVE1a01i
with AscI/SpeI and ligating the two together. The final shuttle
plasmid, pAVE1a04i, which has E1a/E1b under the control of the
tyrosinase promoter, is utilized to make a recombinant virus
identically as described above.
The Colon Cancer-Specific CEA Promoter
[0229] The CEA promoter was cloned from human genomic DNA as
described above and cloned in a similar way into the pAVE1a01i
plasmid using the primers shown in Table 1. The final shuttle
plasmid, pAVE1a05i, is used to generate recombinant virus as
described above.
Replacing the Promoter of E2a on an Adenoviral Vector with a Tumor
Specific Ppromoter
[0230] Constructs made as above will have the E2a protein
(essential for viral replication expressed only in tumor cells.
Therefore, replication of the vector occurs only in tumor cells.
All four of these very specific promoters (in the examples above)
are used to place the E2a coding region obtained from pSE280-E2a
(see U.S. patent application to Kayden et al, "Improved adenoviral
vectors and producer cells" filed Jun. 2, 1995) under the control
of that tumor-specific promoter. The resultant plasmid is
recombined with Add1327, using standard methods of homologous
recombination. The final virus is grown in the cell lines described
in the aforementioned patent application or in the tumor specific
cell lines. The E2a protein, because it is needed in stoichiometric
amounts, has the ability to regulate the degree of replication over
a broad range. This is desirable for therapy. The methods used are
the same as those described for E1a . The difference is that a
shuttle plasmid is used that places E2a under the control of the
tumor specific promoter and returns it to a virus backbone (by
homologous recombination) that has the E2a and E3 genes
deleted.
Replacement of Other Therapeutic Toxic Genes into the
Tumor-Specific Replication Competent Vectors
[0231] Genes such as TK, cytokines, or any therapeutic genes can be
placed into the E3 region of the vector backbone by standard
plasmid construction and homologous recombination. Those genes can
be placed under the control of an E1a-dependent promoter, or a
constitutive promoter such as RSV or CMV.
EXAMPLE 2
[0232] AV5E1aTK01i Construction
[0233] pAVS10TK1 (U.S. application Ser. No. 08/444,284) (the source
of the TK gene in Av15EKa04i below) was digested with XbaI and SpeI
and the resulting 1230 bp fragment, containing the HSV-TK open
reading frame, was ligated into the partially digested XbaI site at
base pair position 6162 of prepac (FIG. 3A) (described in U.S.
patent application Ser. No. 08/852,924). This shuttle plasmid
contains the last 8886 base pairs from 25171 through 34057 of the
Add1327 genome (Thimmapaya, Cell 31:543 (1983)) cloned into
pBluescript SKII(+) (Stratagene). Prepac (FIG. 3A) is a large
plasmid that contains the adenoviral genomic DNA from the right ITR
through the E3 region. The TK gene derived from pAVS10TKI was
inserted into the XbaI site in prepac, putting this gene under the
control of the E3 promoter. The resulting ligated plasmid DNA was
transformed into E. coli STBL2 cells (Life Technologies) and
labeled as prepacTK. The plasmid prepacTK was then used to make the
plasmid prepacTKbnl (FIG. 3B) by ligating the following two
annealed oligonucleotides (5'-GGCCGCATGCATGTTTAAACG-3' (SEQ ID NO:
1) and 5'-GATCCGTTTAAACATGCATGC-3' (SEQ ID NO: 2)) into the BamHI
and NotI digested sites of prepacTK (FIGS. 3A, 3B). The ligation of
this oligonucleotide creates another BamHI site without destroying
the open reading frame.
[0234] The plasmid prepacTKm was then cloned by digesting
prepacTKbnl with BamHI and SpeI and isolating the resulting 11148
base pair fragment (FIG. 3B). This removes a portion of the E2a
gene and allows it to be replaced with a modified E2a gene
containing a mutation that permits replication in monkey cells
(from pG1MBS, see below). This 11198 base pair fragment was ligated
to the 5520 base pair fragment obtained by digesting the plasmid
pG1MBS with BamHI and SpeI (FIG. 3B). The final plasmid is
designated prepacTKm. This plasmid has the TK gene in the E3
region, and also contains the E2a mutation as explained below.
[0235] The plasmid pG1MBS contains the adenoviral type 5 (Ad5)
region between the BamHI site and the SpeI site (Ad5 genome base
pair positions 21562 to 27082, respectively, FIG. 3B). Plasmid
pG1MBS has a point mutation in the adenoviral sequences at base
pair 11670 which allows for increased adenoviral replication in
monkeys (as described by Kruijen, Nucl. Acids Res. 9:4439
(1981).
[0236] To prepare the adenoviral vector Av5E1aTK10i the plasmid
pREpacTKm (FIG. 3C) was digested with the restriction enzymes BamHI
and SalI and the 13748 base pair fragment was isolated by agarose
gel electrophoresis. Adenovirus Add1327 DNA was prepared by
digesting cesium density gradient centrifugation purified virus
with Proteinase K. The Proteinase K digested Add1327 viral DNA was
then extracted first with phenol/chloroform followed by chloroform
and finally with buffer saturated ether. The DNA was recovered
after equlibrating in water using an Amicon Centricon 100 unit. The
purified Add1327 DNA was then digested with BamHI and the 21562
base pair fragment was isolated by agarose gel electrophoresis. The
13748 base pair BamHI/SalI fragment from pREpacTKm and the 21562
base pair Bam HI fragment from Add1327 were then ligated together.
The resulting ligated fragments were then transfected into
A549.A30.S8 cells using Lipofectamine (Life Technologies Inc.) and
incubated at 37.degree. C. in a 5% CO.sub.2, humidified, incubator
until cytopathic effects (CPE) were observed. The resulting
recombinant Av5E1aTK10i (FIG. 3C) virus was then plaque purified on
A549.A30.S8 cells. Av5E1aTK10i plaques were screened by PCR for the
presence of HSV-TK sequences using the following primers: (LMC11:
5'-AGCAAGAAGCCACGG AAGTC-3' (SEQ ID NO: 3) and LMC12:
5'-AGGTCGCAGATCGTCGGTAT-3' (SEQ ID NO: 4)).
Av15E1a04i Construction
[0237] Av1E1a04i was completely digested with SrfI (FIG. 4A)
confirmed digestion on 1% agarose gel in 1.times. TAE, organic
extracted digest with buffered phenol, buffered
phenol/chloroform/isoamyl alcohol (25:24:1), and chloroform, and
then added 1/10 volume 3M NaOAc and 2.5 volume 95% EtOH to
precipitate. The precipitate was pelleted by 20 minute
centrifugation at 12000 g, washed 1.times. with 70% EtOH, and then
air dried for 30 minutes. The pellet was resuspended in dH.sub.2O
and digested with BamHI. The complete digestion was confirmed and
purified as before.
[0238] 293 cells were cotransfected with 2.5 ug Av5E1aTk01i
(digested with Bst 1107 and ClaI (FIG. 4B) and 2.5 ug Av1E1a04i
(digested with Bam HI and SifI, FIG. 4A) using Promega CaCl.sub.2
Transfection Kit (FIG. 4C). Transfections were washed 1.times. with
Richters media supplemented with 10% FBS (R10) and then overlaid
with 1.times. MEM/10% FBS/0.5 % Pen-Strep/1% Fungizone. Plaques
were identified by microscopic examination and picked with 1000 ul
pipet tip into 500 ul R5 media. Plaques were lysed by freeze-thaw
(4.times.) and then infected into S8 cells stimulated with 0.3 um
dexamethasone. When early CPE was evident, the cells were washed
1.times. with PBS, lysed with 200 ul 1N NaOH and neutralized with
30 ul of 7.5 M ammonium acetate. The CVL was diluted 1:50 in
dH.sub.2O and 5 ul of the diluted lysate was used in a 50 ul PCR
reaction using the BMB master mix reagents.
1 E1a promoter primer pair detects both Av15E1aTk04i (1653 bp) and
Add1327 (405 bp) CH12 5'-GACCGTTTACGTGGAGACTCGC-3' (SEQ ID NO:5) bp
367 in ITR CH13 5'-ACCGCCAACATTACAGAGTC- G-3' (SEQ ID NO:6) bp 772
or bp 2020 in E1a gene of Add1327 or Av15E1aTk04i, re- spectively.
HSV-TK primer pair 990 bp product in either Av15E1aTk04i or
Av5E1aTk01i LMC11 5'-AGCAAGAAGCCACGGAAGTC-3' (SEQ ID NO:3) bp 100
of HSV-Tk ORF LMC12 5'-AGGTCGCAGATCGTCGGTAT-3' (SEQ ID NO:4) bp
1090 of HSV-Tk ORF
[0239] Three primary plaques were identified for additional plaque
purification on S8 cells using same PCR of CVL to identify positive
plaques. One plaque was subsequently purified three times by plaque
purification on S8 cells. A bulk preparation of the tertiary plaque
for Av15E1a Tk04i has a titer of 9.times.10.sup.10 particles/ml,
ratio 26.
[0240] Av15E1aTk04i (FIG. 4C) has the AFP promoter controlling E1a
expression, and thus, only AFP positive cells are permissive for
vector replication. Av15E1aTk04i also has an E2a mutation (hr404)
which makes monkey cells permissive for replication, potentially
expanding the range of permissive species to additional restricted
species. Finally, Av15E1aTk)4i has HSV-TK under the control of the
E3 promoter which is positively regulated by E1a; therefore, there
should be no expression in the absence of E1a, which will only be
present in AFP positive cells. As a safety feature in the event
that replication occurs in nontarget cells, then only the nontarget
and target cells that are replicating vector would be sensitive to
GCV treatment. Any infected, nontarget cells that are not
replicating vector will be insensitive to GCV.
EXAMPLE 3
[0241] Av15E1aTk04i should have the same tissue specific
replication restricted adenoviral profile as Av1E1a04i,
specifically, replication only in AFP positive cells (as described
in PCT U.S. application Ser. No. 95/15455 (U.S. Pat. No.
5,998,205)). Infection of Hep3B and HepG2 (AFP positive
hepatocellular carcinoma cell lines) and S8 (E1a positive cell
line) with Add1327, Av1E1a04i, or Av15E1aTk04i results in visible
CPE at infections of 0.5, 5, and 50 particle per cell (FIG. 5).
Av1nBg02v (E1 deleted virus) infection at the same input particle
numbers exhibit no visible CPE except in S8 cells. Conversely,
Chang and HeLa (AFP negative cell lines) exhibit visible CPE only
when infected with Add1327 (wild type) when infected at the same
input particles. Av1E1a04i, Av15E1aTk04i, and Av1nBg02v all
manifest no CPE at infections of 0.5, 5, and 50 particle per cell
in Chang and HeLa cells (FIG. 5). This indicates that AV15E1aTK04i
also replicates specifically in AFP-positive cell lines. Therefore,
the E1a gene product, which controls the E3 promoter, must also be
induced specifically in AFP-positive cell lines. Therefore, HSV-TK,
which is controlled by the E3 promoter, is also induced
specifically in AFP-positive cell lines.
EXAMPLE 4
[0242] Replacement of Other Therapeutic Toxic Genes into the
Tumor-Specific Replication Competent Vectors
[0243] Genes such as TK, cytokines, or any therapeutic genes can be
placed into the E3 region of the vector backbone by standard
plasmid construction and homologous recombination. Those genes can
be placed under the control of an E1a-dependent promoter, or a
constitutive promoter such as RSV or CMV. In vitro control of
replication by inclusion of HSV-TK into the E3 region (FIG. 6).
[0244] HSV-TK was placed into the E3 region of Add1327 to form
Av5E1aTK01i. A549 cells were transduced with either Add1327 or
Av5E1aTK01i. Cells were then treated with 10 .mu.M GCV for 5 days.
Following the 5 day incubation cells were harvested, washed in
HBSS, and then freeze/thawed to prepare a crude viral lysate.
Titers were then performed by standard TCID.sup.50 assays on 293
cells to determine viral titer. The experiment was repeated three
times. Results show that while GCV had no effect on an adenovirus
not carrying the HSV-TK gene, it caused a greater than 4 order of
magnitude drop in titer of a vector carrying the HSV-TK gene.
[0245] In vivo control of replication by inclusion of HSV-TK into
E3 region (FIG. 7).
[0246] Subcutaneous tumors were formed by injecting
1.times.10.sup.7 A549 cells into the subcutaneous space of the
right flank of nude mice. After tumors formed, 1.times.10.sup.9 pfu
of Av5E1aTK01i was injected into several animals containing tumors.
After 5 days half the animals received 5 days of IP GCV treatment
at 75 mg/kg once a day. At the end of this time frame all animals
were sacrificed. Immunohistochemistry was performed on all samples
for HSV-TK expression and hexon expression, the latter which is
only expressed when the vector is replicating. Results show as seen
in the figure that only hexon is severely diminished when the
animals were treated with GCV.
[0247] The disclosures of all patents, publications (including
published patent applications), and database accession numbers
referred to in this specification are specifically incorporated
herein by reference in their entirety to the same extent as if each
such individual patent, publication, and database accession numbers
were specifically and individually indicated to be incorporated by
reference in its entirety.
2TABLE 1 Oligonucleotide Primers for Constructing Tissue-Specific
Promoters 1. DF3 (BREAST CANCER) 5' GGG CGC GCC CTG GAA AGT CCG GCT
GGG GCG GGG ACT GTG GGT TTC AGG GTA GAA CTG CGT GTG GAA 3' (SEQ ID
NO:7) 5' CGG GAC AGG GAG CGG TTA GAA GGG TGG GGC TAT TCC GGG AAG
TGG TGG GGG GAG GGA ACT AGT A 3' (SEQ ID NO:8) 5' GAT CTA CTA GTT
CCC TCC CCC CAC CAC TTC CCG GAA TAG CCC CAC CCT TCT AAC CCC TCC CTG
3' (SEQ ID NO:9) 5' TCC CGT TCC ACA CGC ACT TCT ACC CTG AAA CCC ACA
GTC CCC GCC CCA GCC GGA CTT TCC AGG GCG CGC CC 3' (SEQ ID NO:10) 2.
TYROSINASE (MELANOMA) 5' GAC CCG GGC GCG CCG GAG CAG TGC TAT TCA
AAC CAT CCA C 3' (SEQ ID NO:11) 5' CGA GAT CTA CTA GTT CTG CAC CAA
TAG GTT AAT GAG TGT C 3' (SEQ ID NO:12) 3. CEA PROMOTER
(HEPATOCELLULAR CARCINOMA) 5' GAC CCG GGC GCG CCT CTG TCA CCT TCC
TGT TGG 3' (SEQ ID NO:13) 5' CGA GAT CTA CTA GTT CTC TGC TGT CTG
CTC TGT C 3' (SEQ ID NO:14)
[0248]
Sequence CWU 1
1
14 1 21 DNA Artificial Sequence Description of Artificial Sequence
synthetic oligonucleotide 1 ggccgcatgc atgtttaaac g 21 2 21 DNA
Artificial Sequence Description of Artificial Sequence synthetic
oligonucleotide 2 gatccgttta aacatgcatg c 21 3 20 DNA Artificial
Sequence Description of Artificial Sequence primer 3 agcaagaagc
cacggaagtc 20 4 20 DNA Artificial Sequence Description of
Artificial Sequence primer 4 aggtcgcaga tcgtcggtat 20 5 22 DNA
Artificial Sequence Description of Artificial Sequence primer 5
gaccgtttac gtggagactc gc 22 6 21 DNA Artificial Sequence
Description of Artificial Sequence primer 6 accgccaaca ttacagagtc g
21 7 66 DNA Artificial Sequence Description of Artificial Sequence
primer 7 gggcgcgccc tggaaagtcc ggctggggcg gggactgtgg gtttcagggt
agaactgcgt 60 gtggaa 66 8 64 DNA Artificial Sequence Description of
Artificial Sequence primer 8 cgggacaggg agcggttaga agggtggggc
tattccggga agtggtgggg ggagggaact 60 agta 64 9 63 DNA Artificial
Sequence Description of Artificial Sequence primer 9 gatctactag
ttccctcccc ccaccacttc ccggaatagc cccacccttc taaccgctcc 60 ctg 63 10
71 DNA Artificial Sequence Description of Artificial Sequence
primer 10 tcccgttcca cacgcagttc taccctgaaa cccacagtcc ccgccccagc
cggactttcc 60 agggcgcgcc c 71 11 40 DNA Artificial Sequence
Description of Artificial Sequence primer 11 gacccgggcg cgccggagca
gtgctattca aaccatccag 40 12 40 DNA Artificial Sequence Description
of Artificial Sequence primer 12 cgagatctac tagttctgca ccaataggtt
aatgagtgtc 40 13 33 DNA Artificial Sequence Description of
Artificial Sequence primer 13 gacccgggcg cgcctctgtc accttcctgt tgg
33 14 34 DNA Artificial Sequence Description of Artificial Sequence
primer 14 cgagatctac tagttctctg ctgtctgctc tgtc 34
* * * * *