U.S. patent application number 12/118008 was filed with the patent office on 2008-09-25 for method for propagating adenoviral vectors encoding inhibitory gene products.
This patent application is currently assigned to GENVEC, INC.. Invention is credited to Douglas E. Brough, Jason G. D. Gall, C. Richter King.
Application Number | 20080233650 12/118008 |
Document ID | / |
Family ID | 38189139 |
Filed Date | 2008-09-25 |
United States Patent
Application |
20080233650 |
Kind Code |
A1 |
Gall; Jason G. D. ; et
al. |
September 25, 2008 |
Method for propagating adenoviral vectors encoding inhibitory gene
products
Abstract
The invention provides a method of propagating an adenoviral
vector. The method comprises (a) providing a cell comprising a
cellular genome comprising a nucleic acid sequence encoding a
tetracycline operon repressor protein (tetR), and (b) contacting
the cell with an adenoviral vector comprising a heterologous
nucleic acid sequence encoding a toxic protein. The heterologous
nucleic acid sequence is operably linked to a promoter and one or
more tetracycline operon operator sequences (tetO), and expression
of the heterologous nucleic acid sequence is inhibited in the
presence of tetR, such that the adenoviral vector is propagated.
The invention also provides a system comprising the aforementioned
cell and adenoviral vector.
Inventors: |
Gall; Jason G. D.;
(Germantown, MD) ; Brough; Douglas E.;
(Gaithersburg, MD) ; King; C. Richter;
(Washington, DC) |
Correspondence
Address: |
LEYDIG VOIT & MAYER, LTD
TWO PRUDENTIAL PLAZA, SUITE 4900, 180 NORTH STETSON AVENUE
CHICAGO
IL
60601-6731
US
|
Assignee: |
GENVEC, INC.
Gaithersburg
MD
|
Family ID: |
38189139 |
Appl. No.: |
12/118008 |
Filed: |
May 9, 2008 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
PCT/US2006/060732 |
Nov 8, 2006 |
|
|
|
12118008 |
|
|
|
|
60735578 |
Nov 10, 2005 |
|
|
|
Current U.S.
Class: |
435/456 ;
435/325 |
Current CPC
Class: |
C12N 15/86 20130101;
C12N 2830/006 20130101; C12N 7/00 20130101; C12N 2710/10352
20130101; C12N 2710/10343 20130101; C12N 15/635 20130101 |
Class at
Publication: |
435/456 ;
435/325 |
International
Class: |
C12N 15/87 20060101
C12N015/87; C12N 5/06 20060101 C12N005/06 |
Claims
1. A method of propagating an adenoviral vector, which method
comprises: (a) providing a cell comprising a cellular genome
comprising a nucleic acid sequence encoding a tetracycline operon
repressor protein (tetR), and (b) contacting the cell with an
adenoviral vector having an adenoviral genome comprising a
heterologous nucleic acid sequence encoding a protein that is toxic
to the cell, wherein the heterologous nucleic acid sequence is
operably linked to a promoter and one or more tetracycline operon
operator sequences (tetO), so as to transfect the cell with the
adenoviral vector, wherein the nucleic acid sequence encoding tetR
is expressed to produce tetR, expression of the heterologous
nucleic acid sequence is inhibited in the presence of tetR, and the
adenoviral vector is propagated.
2. The method of claim 1, wherein the adenoviral vector is
replication-deficient.
3. The method of claim 2, wherein the adenoviral vector requires,
at most, complementation of the E1 region of the adenoviral genome
for replication.
4. The method of claim 2, wherein the adenoviral vector requires,
at most, complementation of the E4 region of the adenoviral genome
for replication.
5. The method of claim 2, wherein the adenoviral vector has an
adenoviral genome devoid of all of the E1 region and at least a
portion of the E4 region, and the adenoviral vector requires, at
most, complementation of the E1 and E4 regions of the adenoviral
genome for replication.
6. The method of claim 1 wherein the adenoviral vector lacks all or
part of the E3 region of the adenoviral genome.
7. The method of claim 1, wherein the adenoviral genome is a
subgroup C adenoviral genome.
8. The method of claim 1, wherein the adenoviral genome is a
non-subgroup C adenoviral genome.
9. The method of claim 1, wherein the heterologous nucleic acid
sequence encodes an env, gag, or pol protein from clades A, B, or C
of a human immunodeficiency virus (HIV), or a fusion protein
comprising any of the foregoing.
10. The method of claim 1, wherein the heterologous nucleic acid
sequence encodes an E protein, an M protein, or a spike protein of
a severe acute respiratory syndrome (SARS) virus.
11. The method of claim 1, wherein the heterologous nucleic acid
sequence encodes a transforming growth factor .beta. (TGF.beta.),
an antibiotic, a malaria protein, or a nitric oxide synthase.
12. The method of claim 1, wherein the cell comprises a cellular
genome into the nuclear genome of which is inserted the open
reading frame-6 (ORF-6) and no other open reading frame of the E4
region of an adenoviral genome operably linked to a promoter, and
which cell line complements in trans an adenoviral vector
comprising an adenoviral genome having a deletion of the E1 and E4
regions of the adenoviral genome.
13. The method of claim 12, wherein the ORF-6 of the E4 region of
the adenoviral genome is operably linked to an inducible
promoter.
14. A system comprising: (a) a cell comprising a cellular genome
comprising a nucleic acid sequence encoding a tetracycline operon
repressor protein (tetR), which can be expressed to produce tetR,
and (b) an adenoviral vector having an adenoviral genome comprising
a heterologous nucleic acid sequence encoding a protein that is
toxic to the cell, wherein the heterologous nucleic acid sequence
is operably linked to a promoter and one or more tetracycline
operon operator sequences (tetO), and wherein the adenoviral vector
can transfect the cell and be propagated in the cell.
15. The system of claim 14, wherein the adenoviral vector is
replication-deficient.
16. The system of claim 15, wherein the adenoviral vector requires,
at most, complementation of the E1 region of the adenoviral genome
for replication.
17. The system of claim 15 or claim 16, wherein the adenoviral
vector requires, at most, complementation of the E4 region of the
adenoviral genome for replication.
18. The system of claim 15, wherein the adenoviral vector has an
adenoviral genome devoid of all of the E1 region and at least a
portion of the E4 region, and the adenoviral vector requires, at
most, complementation of the E1 and E4 regions of the adenoviral
genome for replication.
19. The system of claim 14, wherein the adenoviral vector lacks all
or part of the E3 region of the adenoviral genome.
20. The system of claim 14, wherein the adenoviral genome is a
subgroup C adenoviral genome.
21. The system of claim 14, wherein the adenoviral genome is a
non-subgroup C adenoviral genome.
22. The system claim 14, wherein the heterologous nucleic acid
sequence encodes an env, gag, or pol protein from clades A, B, or C
of a human immunodeficiency virus (HIV), or a fusion protein
comprising any of the foregoing.
23. The system of claim 14, wherein the heterologous nucleic acid
sequence encodes an E protein, an M protein, or a spike protein of
a severe acute respiratory syndrome (SARS) virus.
24. The system of claim 14, wherein the heterologous nucleic acid
sequence encodes a transforming growth factor .beta. (TGF.beta.),
an antibiotic, a malaria protein, or a nitric oxide synthase.
25. The system of claim 14, wherein the cell comprises a cellular
genome into the nuclear genome of which is inserted the open
reading frame-6 (ORF-6) and no other open reading frame of the E4
region of an adenoviral genome operably linked to a promoter, and
which cell line complements in trans an adenoviral vector
comprising an adenoviral genome having a deletion of the E1 and E4
regions of the adenoviral genome.
26. The system of claim 25, wherein the ORF-6 of the E4 region of
the adenoviral genome is operably linked to an inducible
promoter.
27. The method of claim 1, wherein the adenoviral vector is
replication-competent.
28. The method of claim 1, wherein the heterologous nucleic acid
sequence encodes protein 1A, 1B, 1C, 1D, 2A, 2B, 2C, 3A, 3B, 3C, or
3D of a foot-and-mouth disease virus (FMD).
29. The system of claim 16, wherein the adenoviral vector is
replication-competent.
31. The system of claim 16, wherein the heterologous nucleic acid
sequence encodes protein 1A, 1B, 1C, 1D, 2A, 2B, 2C, 3A, 3B, 3C, or
3D of a foot-and-mouth disease virus (FMD).
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This patent application is a continuation of copending
International Patent Application No. PCT/US2006/060732, filed Nov.
8, 2006, designating the United States, which claims the benefit of
U.S. Provisional Patent Application No. 60/735,578, filed Nov. 10,
2005.
INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY
[0002] Incorporated by reference in its entirety herein is a
computer-readable nucleotide/amino acid sequence listing submitted
concurrently herewith and identified as follows: One 2,357 Byte
ASCII (Text) file named "702966_ST25.TXT," created on May 7,
2008.
BACKGROUND OF THE INVENTION
[0003] Delivery of proteins as therapeutics or for inducing an
immune response in biologically relevant amounts has been an
obstacle to drug and vaccine development for decades. One solution
that has proven to be a successful alternative to traditional drug
delivery approaches is delivery of exogenous nucleic acid sequences
for production of therapeutic factors in vivo. Gene transfer
vectors ideally enter a wide variety of cell types, have the
capacity to accept large nucleic acid sequences, are safe, and can
be produced in quantities required for treating patients. Viral
vectors have these advantageous properties and are used in a
variety of protocols to treat or prevent biological disorders.
[0004] Adenoviral vectors are attractive for gene transfer
applications, such as gene therapy and vaccines as a result of
their ability to infect a variety of cell types with high
efficiency. Adenoviral vectors containing a heterologous transgene
under the control of astrong promoter are potent, achieving
expression of the heterologous protein up to 20% of total cell
proteins (see, e.g., Massie et al., J. Virol., 72, 2289-2296
(1998)). A high level of transgene expression, however, often is
inhibitory to virus growth, such as when the transgene encodes a
protein that is cytotoxic to a packaging cell. Thus, high
expression of an adenovirus-encoded transgene can prevent the
production of viable adenoviral vector particles from naked DNA
(see, e.g., Matthews et al., J. Gen. Virol., 80 (Pt 2), 345-353
(1999)), or reduce the productivity of virus growth within
packaging cells (see, e.g., Molin et al., J. Virol., 74, 9002-9009
(2000)).
[0005] To better regulate transgene expression within virus
packaging cells while maintaining vector potency, gene regulation
systems have been employed in the construction of adenoviral
vectors. These systems typically incorporate transcriptional
regulatory proteins into the adenoviral vector or in the target
cell (see, e.g., Massie et al., supra, Goukassian et al., FASEB J,
15, 1877-1885 (2001), Mizuguchi et al., Biochim. Biophys. Acta,
1568, 21-29 (2001), Rubinchik et al., Mol. Ther., 4, 416-426
(2001), Molin et al., J. Virol., 72, 8358-8361 (1998), Hu et al.,
Cancer Res., 57, 3339-3343 (1997), Edholm et al., J. Virol., 75,
9579-9584 (2001), and U.S. Pat. No. 6,391,612). Such gene
regulation systems, however, often require the use of inducer
compounds (e.g., tetracycline analogs), which increases the time
required to generate viable adenoviral vector particles, thereby
complicating their widespread use.
[0006] Therefore, there remains a need for more efficient methods
of propagating adenoviral vectors encoding transgenes whose
expression inhibits viral growth in host cells. The invention
provides such a method. These and other advantages of the
invention, as well as additional inventive features, will be
apparent from the description of the invention provided herein.
BRIEF SUMMARY OF THE INVENTION
[0007] The invention provides a method of propagating an adenoviral
vector, which method comprises (a) providing a cell comprising a
cellular genome comprising a nucleic acid sequence encoding a
tetracycline operon repressor protein (tetR), and (b) contacting
the cell with an adenoviral vector having an adenoviral genome
comprising a heterologous nucleic acid sequence encoding a protein
that is toxic to the cell, wherein the heterologous nucleic acid
sequence is operably linked to a promoter and one or more
tetracycline operon operator sequences (tetO), so as to transfect
the cell with the adenoviral vector. The nucleic acid sequence
encoding tetR is expressed to produce tetR, expression of the
heterologous nucleic acid sequence is inhibited in the presence of
tetR, and the adenoviral vector is propagated.
[0008] The invention also provides a system comprising (a) a cell
comprising a cellular genome comprising a nucleic acid sequence
encoding a tetracycline operon repressor protein (tetR), which can
be expressed to produce tetR, and (b) an adenoviral vector having
an adenoviral genome comprising a heterologous nucleic acid
sequence encoding a protein that is toxic to the cell. The
heterologous nucleic acid sequence is operably linked to a promoter
and one or more tetracycline operon operator sequences (tetO), and
the adenoviral vector can transfect the cell and be propagated in
the cell.
DETAILED DESCRIPTION OF THE INVENTION
[0009] The invention is predicated, at least in part, on the
discovery that adenoviral vectors encoding inhibitory gene products
can be produced using a tetracycline operon-based gene regulation
system in which regulation of gene expression is mediated through
the packaging cell line, wherein the addition of inducer compounds
is not required.
[0010] The invention provides a method of propagating an adenoviral
vector. Adenovirus from various origins, subtypes, or mixture of
subtypes can be used as the source of the viral genome for the
adenoviral vector. While non-human adenovirus (e.g., simian, avian,
canine, ovine, or bovine adenoviruses) can be used to generate the
adenoviral vector, a human adenovirus preferably is used as the
source of the viral genome for the adenoviral vector of the
inventive method. Adenovirus can be of various subgroups or
serotypes. For instance, an adenovirus can be of subgroup A (e.g.,
serotypes 12, 18, and 31), subgroup B (e.g., serotypes 3, 7, 11,
14, 16, 21, 34, 35, and 50), subgroup C (e.g., serotypes 1, 2, 5,
and 6), subgroup D (e.g., serotypes 8, 9, 10, 13, 15, 17, 19, 20,
22-30, 32, 33, 36-39, and 42-48), subgroup E (e.g., serotype 4),
subgroup F (e.g., serotypes 40 and 41), an unclassified serogroup
(e.g., serotypes 49 and 51), or any other adenoviral serotype.
Adenoviral serotypes 1 through 51 are available from the American
Type Culture Collection (ATCC, Manassas, Va.). Preferably, in the
context of the inventive method, the adenoviral vector is of human
subgroup C, especially serotype 2 or even more desirably serotype
5. However, non-group C adenoviruses can be used to prepare
adenoviral gene transfer vectors for delivery of gene products to
host cells. Preferred adenoviruses used in the construction of
non-group C adenoviral gene transfer vectors include Ad12 (group
A), Ad7 and Ad35 (group B), Ad30 and Ad36 (group D), Ad4 (group E),
and Ad41 (group F). Non-group C adenoviral vectors, methods of
producing non-group C adenoviral vectors, and methods of using
non-group C adenoviral vectors are disclosed in, for example, U.S.
Pat. Nos. 5,801,030, 5,837,511, and 5,849,561 and International
Patent Applications WO 97/12986 and WO 98/53087.
[0011] The adenoviral vector can comprise a mixture of subtypes and
thereby be a "chimeric" adenoviral vector. A chimeric adenoviral
vector can comprise an adenoviral genome that is derived from two
or more (e.g., 2, 3, 4, etc.) different adenovirus serotypes. In
the context of the invention, a chimeric adenoviral vector can
comprise approximately equal amounts of the genome of each of the
two or more different adenovirus serotypes. When the chimeric
adenoviral vector genome is comprised of the genomes of two
different adenovirus serotypes, the chimeric adenoviral vector
genome preferably comprises no more than about 70% (e.g., no more
than about 65%, about 50%, or about 40%) of the genome of one of
the adenovirus serotypes, with the remainder of the chimeric
adenovirus genome being derived from the genome of the other
adenovirus serotype. In one embodiment, the chimeric adenoviral
vector can contain an adenoviral genome comprising a portion of a
serotype 2 genome and a portion of a serotype 5 genome. For
example, nucleotides 1-456 of such an adenoviral vector can be
derived from a serotype 2 genome, while the remainder of the
adenoviral genome can be derived from a serotype 5 genome.
[0012] The adenoviral vector of the invention can be
replication-competent. For example, the adenoviral vector can have
a mutation (e.g., a deletion, an insertion, or a substitution) in
the adenoviral genome that does not inhibit viral replication in
host cells. The inventive adenoviral vector also can be
conditionally replication-competent. Preferably, however, the
adenoviral vector is replication-deficient in host cells.
[0013] By "replication-deficient" is meant that the adenoviral
vector requires complementation of one or more regions of the
adenoviral genome that are required for replication, as a result
of, for example a deficiency in at least one replication-essential
gene function (i.e., such that the adenoviral vector does not
replicate in typical host cells, especially those in a human
patient that could be infected by the adenoviral vector in the
course of the inventive method). A deficiency in a gene, gene
function, gene, or genomic region, as used herein, is defined as a
deletion of sufficient genetic material of the viral genome to
obliterate or impair the function of the gene (e.g., such that the
function of the gene product is reduced by at least about 2-fold,
5-fold, 10-fold, 20-fold, 30-fold, or 50-fold) whose nucleic acid
sequence was deleted in whole or in part. Deletion of an entire
gene region often is not required for disruption of a
replication-essential gene function. However, for the purpose of
providing sufficient space in the adenoviral genome for one or more
transgenes, removal of a majority of a gene region may be
desirable. While deletion of genetic material is preferred,
mutation of genetic material by addition or substitution also is
appropriate for disrupting gene function. Replication-essential
gene functions are those gene functions that are required for
replication (e.g., propagation) and are encoded by, for example,
the adenoviral early regions (e.g., the E1, E2, and E4 regions),
late regions (e.g., the L1-L5 regions), genes involved in viral
packaging (e.g., the IVa2 gene), and virus-associated RNAs (e.g.,
VA-RNA1 and/or VA-RNA-2).
[0014] The replication-deficient adenoviral vector desirably
requires complementation of at least one replication-essential gene
function of one or more regions of the adenoviral genome.
Preferably, the adenoviral vector requires complementation of at
least one gene function of the E1A region, the E1B region, or the
E4 region of the adenoviral genome required for viral replication
(denoted an E1-deficient or E4-deficient adenoviral vector). In
addition to a deficiency in the E1 region, the recombinant
adenovirus also can have a mutation in the major late promoter
(MLP), as discussed in International Patent Application Publication
WO 00/00628. Most preferably, the adenoviral vector is deficient in
at least one replication-essential gene function (desirably all
replication-essential gene functions) of the E1 region and at least
one gene function of the nonessential E3 region (e.g., an Xba I
deletion of the E3 region) (denoted an E1/E3-deficient adenoviral
vector). With respect to the E1 region, the adenoviral vector can
be deficient in part or all of the E1A region and/or part or all of
the E1B region, e.g., in at least one replication-essential gene
function of each of the E1A and E1B regions, thus requiring
complementation of the E1A region and the E1B region of the
adenoviral genome for replication. The adenoviral vector also can
require complementation of the E4 region of the adenoviral genome
for replication, such as through a deficiency in one or more
replication-essential gene functions of the E4 region.
[0015] When the adenoviral vector is E1-deficient, the adenoviral
vector genome can comprise a deletion beginning at any nucleotide
between nucleotides 335 to 375 (e.g., nucleotide 356) and ending at
any nucleotide between nucleotides 3,310 to 3,350 (e.g., nucleotide
3,329) or even ending at any nucleotide between 3,490 and 3,530
(e.g., nucleotide 3,510) (based on the adenovirus serotype 5
genome).
[0016] When E2A-deficient, the adenoviral vector genome can
comprise a deletion beginning at any nucleotide between nucleotides
22,425 to 22,465 (e.g., nucleotide 22,443) and ending at any
nucleotide between nucleotides 24,010 to 24,050 (e.g., nucleotide
24,032) (based on the adenovirus serotype 5 genome). When
E3-deficient, the adenoviral vector genome can comprise a deletion
beginning at any nucleotide between nucleotides 28,575 to 29,615
(e.g., nucleotide 28,593) and ending at any nucleotide between
nucleotides 30,450 to 30,490 (e.g., nucleotide 30,470) (based on
the adenovirus serotype 5 genome).
[0017] When the adenoviral vector is deficient in at least one
replication-essential gene function in one region of the adenoviral
genome (e.g., an E1- or E1/E3-deficient adenoviral vector), the
adenoviral vector is referred to as "singly replication-deficient."
A particularly preferred singly replication-deficient adenoviral
vector is, for example, a replication-deficient adenoviral vector
requiring, at most, complementation of the E1 region of the
adenoviral genome, so as to propagate the adenoviral vector (e.g.,
to form adenoviral vector particles).
[0018] The adenoviral vector of the invention can be "multiply
replication-deficient," meaning that the adenoviral vector is
deficient in one or more replication-essential gene functions in
each of two or more regions of the adenoviral genome, and requires
complementation of those functions for replication. For example,
the aforementioned E1-deficient or E1/E3-deficient adenoviral
vector can be further deficient in at least one
replication-essential gene function of the E4 region (denoted an
E1/E4- or E1/E3/E4-deficient adenoviral vector), and/or the E2
region (denoted an E1/E2- or E1/E2/E3-deficient adenoviral vector),
preferably the E2A region (denoted an E1/E2A- or
E1/E2A/E3-deficient adenoviral vector). An adenoviral vector
deleted of the entire E4 region can elicit a lower host immune
response. When E4-deficient, the adenoviral vector genome can
comprise a deletion beginning at, for example, any nucleotide
between nucleotides 32,805 to 32,845 (e.g., nucleotide 32,826) and
ending at, for example, any nucleotide between nucleotides 35,540
to 35,580 (e.g., nucleotide 35,561) (based on the adenovirus
serotype 5 genome), optionally in addition to deletions in the E1
region (e.g., nucleotides 356 to 3,329 or nucleotides 356 to 3,510)
(based on the adenovirus serotype 5 genome) and/or deletions in the
E3 region (e.g., nucleotides 28,594 to 30,469 or nucleotides 28,593
to 30,470) (based on the adenovirus serotype 5 genome). The
endpoints defining the deleted nucleotide portions can be difficult
to precisely determine and typically will not significantly affect
the nature of the adenoviral vector, i.e., each of the
aforementioned nucleotide numbers can be +/-1, 2, 3, 4, 5, or even
10 or 20 nucleotides.
[0019] If the adenoviral vector of the invention is deficient in a
replication-essential gene function of the E2A region, the vector
preferably does not comprise a complete deletion of the E2A region,
which deletion preferably is less than about 230 base pairs in
length. Generally, the E2A region of the adenovirus codes for a DBP
(DNA binding protein), a polypeptide required for DNA replication.
DBP is composed of 473 to 529 amino acids depending on the viral
serotype. It is believed that DBP is an asymmetric protein that
exists as a prolate ellipsoid consisting of a globular Ct with an
extended Nt domain. Studies indicate that the Ct domain is
responsible for DBP's ability to bind to nucleic acids, bind to
zinc, and function in DNA synthesis at the level of DNA chain
elongation. However, the Nt domain is believed to function in late
gene expression at both transcriptional and post-transcriptional
levels, is responsible for efficient nuclear localization of the
protein, and also may be involved in enhancement of its own
expression. Deletions in the Nt domain between amino acids 2 to 38
have indicated that this region is important for DBP function
(Brough et al., Virology, 196, 269-281 (1993)). While deletions in
the E2A region coding for the Ct region of the DBP have no effect
on viral replication, deletions in the E2A region which code for
amino acids 2 to 38 of the Nt domain of the DBP impair viral
replication. It is preferable that any multiply
replication-deficient adenoviral vector contains this portion of
the E2A region of the adenoviral genome. In particular, for
example, the desired portion of the E2A region to be retained is
that portion of the E2A region of the adenoviral genome which is
defined by the 5' end of the E2A region, specifically positions
Ad5(23816) to Ad5(24032) of the E2A region of the adenoviral genome
of serotype Ad5. This portion of the adenoviral genome desirably is
included in the adenoviral vector because it is not complemented in
current E2A cell lines so as to provide the desired level of viral
propagation.
[0020] While the above-described deletions are described with
respect to an adenovirus serotype 5 genome, one of ordinary skill
in the art can determine the nucleotide coordinates of the same
regions of other adenovirus serotypes, such as an adenovirus
serotype 2 genome, without undue experimentation, based on the
similarity between the genomes of various adenovirus serotypes,
particularly adenovirus serotypes 2 and 5.
[0021] In one embodiment of the inventive method, the adenoviral
vector can comprise an adenoviral genome deficient in one or more
replication-essential gene functions of each of the E1 and E4
regions (i.e., the adenoviral vector is an E1/E4-deficient
adenoviral vector), preferably with the entire coding region of the
E4 region having been deleted from the adenoviral genome. In other
words, all the open reading frames (ORFs) of the E4 region have
been removed. Most preferably, the adenoviral vector is rendered
replication-deficient by deletion of all of the E1 region and by
deletion of a portion of the E4 region. The E4 region of the
adenoviral vector can retain the native E4 promoter,
polyadenylation sequence, and/or the right-side inverted terminal
repeat (ITR).
[0022] It should be appreciated that the deletion of different
regions of the adenoviral vector can alter the immune response of
the mammal. In particular, deletion of different regions can reduce
the inflammatory response generated by the adenoviral vector.
Furthermore, the adenoviral vector's coat protein can be modified
so as to decrease the adenoviral vector's ability or inability to
be recognized by a neutralizing antibody directed against the
wild-type coat protein, as described in International Patent
Application WO 98/40509. Such modifications are useful for
long-term treatment of persistent ocular disorders.
[0023] The adenoviral vector, when multiply replication-deficient,
especially in replication-essential gene functions of the E1 and E4
regions, can include a spacer sequence to provide viral growth in a
complementing cell line similar to that achieved by singly
replication-deficient adenoviral vectors, particularly an
E1-deficient adenoviral vector. In a preferred E4-deficient
adenoviral vector of the invention wherein the L5 fiber region is
retained, the spacer is desirably located between the L5 fiber
region and the right-side ITR. More preferably in such an
adenoviral vector, the E4 polyadenylation sequence alone or, most
preferably, in combination with another sequence exists between the
L5 fiber region and the right-side ITR, so as to sufficiently
separate the retained L5 fiber region from the right-side ITR, such
that viral production of such a vector approaches that of a singly
replication-deficient adenoviral vector, particularly a singly
replication-deficient E1 deficient adenoviral vector.
[0024] The spacer sequence can contain any nucleotide sequence or
sequences which are of a desired length, such as sequences at least
about 15 base pairs (e.g., between about 15 base pairs and about
12,000 base pairs), preferably about 100 base pairs to about 10,000
base pairs, more preferably about 500 base pairs to about 8,000
base pairs, even more preferably about 1,500 base pairs to about
6,000 base pairs, and most preferably about 2,000 to about 3,000
base pairs in length. The spacer sequence can be coding or
non-coding and native or non-native with respect to the adenoviral
genome, but does not restore the replication-essential function to
the deficient region. The spacer can also contain a
promoter-variable expression cassette. More preferably, the spacer
comprises an additional polyadenylation sequence and/or a passenger
gene. Preferably, in the case of a spacer inserted into a region
deficient for E4, both the E4 polyadenylation sequence and the E4
promoter of the adenoviral genome or any other (cellular or viral)
promoter remain in the vector. The spacer is located between the E4
polyadenylation site and the E4 promoter, or, if the E4 promoter is
not present in the vector, the spacer is proximal to the right-side
ITR. The spacer can comprise any suitable polyadenylation sequence.
Examples of suitable polyadenylation sequences include synthetic
optimized sequences, BGH (Bovine Growth Hormone), polyoma virus, TK
(Thymidine Kinase), EBV (Epstein Barr Virus) and the
papillomaviruses, including human papillomaviruses and BPV (Bovine
Papilloma Virus). Preferably, particularly in the E4 deficient
region, the spacer includes an SV40 polyadenylation sequence. The
SV40 polyadenylation sequence allows for higher virus production
levels of multiply replication deficient adenoviral vectors. In the
absence of a spacer, production of fiber protein and/or viral
growth of the multiply replication-deficient adenoviral vector is
reduced by comparison to that of a singly replication-deficient
adenoviral vector. However, inclusion of the spacer in at least one
of the deficient adenoviral regions, preferably the E4 region, can
counteract this decrease in fiber protein production and viral
growth. Ideally, the spacer is composed of the glucuronidase gene.
The use of a spacer in an adenoviral vector is further described
in, for example, U.S. Pat. No. 5,851,806 and International Patent
Application WO 97/21826.
[0025] It has been observed that an at least E4-deficient
adenoviral vector expresses a transgene at high levels for a
limited amount of time in vivo and that persistence of expression
of a transgene in an at least E4-deficient adenoviral vector can be
modulated through the action of a trans-acting factor, such as HSV
ICP0, Ad pTP, CMV-IE2, CMV-IE86, HIV tat, HTLV-tax, HBV-X, AAV Rep
78, the cellular factor from the U205 osteosarcoma cell line that
functions like HSV ICP0, or the cellular factor in PC12 cells that
is induced by nerve growth factor, among others, as described in
for example, U.S. Pat. Nos. 6,225,113, 6,649,373, and 6,660,521,
and International Patent Application Publication WO 00/34496. In
view of the above, a multiply deficient adenoviral vector (e.g.,
the at least E4-deficient adenoviral vector) or a second expression
vector can comprise a nucleic acid sequence encoding a trans-acting
factor that modulates the persistence of expression of the nucleic
acid sequence. Persistent expression of antigenic DNA can be
desired when generating immune tolerance.
[0026] Desirably, the adenoviral vector requires, at most,
complementation of replication-essential gene functions of the E1,
E2A, and/or E4 regions of the adenoviral genome for replication
(i.e., propagation). However, the adenoviral genome can be modified
to disrupt one or more replication-essential gene functions as
desired by the practitioner, so long as the adenoviral vector
remains deficient and can be propagated using, for example,
complementing cells and/or exogenous DNA (e.g., helper adenovirus)
encoding the disrupted replication-essential gene functions. In
this respect, the adenoviral vector can be deficient in
replication-essential gene functions of only the early regions of
the adenoviral genome, only the late regions of the adenoviral
genome, and both the early and late regions of the adenoviral
genome. Suitable replication-deficient adenoviral vectors,
including singly and multiply replication-deficient adenoviral
vectors, are disclosed in U.S. Pat. Nos. 5,837,511, 5,851,806,
5,994,106, 6,127,175, and 6,482,616; U.S. Patent Application
Publications 2001/0043922 A1, 2002/0004040 A1, 2002/0031831 A1,
2002/0110545 A1, and 2004/0161848 A1; and International Patent
Application Publications WO 94/28152, WO 95/02697, WO 95/16772, WO
95/34671, WO 96/22378, WO 97/12986, WO 97/21826, and WO
03/022311.
[0027] By removing all or part of, for example, the E1, E3, and E4
regions of the adenoviral genome, the resulting adenoviral vector
is able to accept inserts of exogenous nucleic acid sequences while
retaining the ability to be packaged into adenoviral capsids. The
nucleic acid sequence can be positioned in the E1 region, the E3
region, or the E4 region of the adenoviral genome. Indeed, the
nucleic acid sequence can be inserted anywhere in the adenoviral
genome so long as the position does not prevent expression of the
nucleic acid sequence or interfere with packaging of the adenoviral
vector.
[0028] If the adenoviral vector is not replication-deficient,
ideally the adenoviral vector is manipulated to limit replication
of the vector to within a target tissue. The adenoviral vector can
be a conditionally-replicating adenoviral vector, which is
engineered to replicate under conditions pre-determined by the
practitioner. For example, replication-essential gene functions,
e.g., gene functions encoded by the adenoviral early regions, can
be operably linked to an inducible, repressible, or tissue-specific
transcription control sequence, e.g., promoter. In this embodiment,
replication requires the presence or absence of specific factors
that interact with the transcription control sequence. In
autoimmune disease treatment, it can be advantageous to control
adenoviral vector replication in, for instance, lymph nodes, to
obtain continual antigen production and control immune cell
production. Conditionally-replicating adenoviral vectors are
described further in U.S. Pat. No. 5,998,205.
[0029] In addition to modification (e.g., deletion, mutation, or
replacement) of adenoviral sequences encoding replication-essential
gene functions, the adenoviral genome can contain benign or
non-lethal modifications, i.e., modifications which do not render
the adenovirus replication-deficient, or, desirably, do not
adversely affect viral functioning and/or production of viral
proteins, even if such modifications are in regions of the
adenoviral genome that otherwise contain replication-essential gene
functions. Such modifications commonly result from DNA manipulation
or serve to facilitate expression vector construction. For example,
it can be advantageous to remove or introduce restriction enzyme
sites in the adenoviral genome. Such benign mutations often have no
detectable adverse effect on viral functioning. For example, the
adenoviral vector can comprise a deletion of nucleotides 10,594 and
10,595 (based on the adenoviral serotype 5 genome), which are
associated with VA-RNA-1 transcription, but the deletion of which
does not prohibit production of VA-RNA-1.
[0030] Similarly, the coat protein of a viral vector, preferably an
adenoviral vector, can be manipulated to alter the binding
specificity or recognition of a virus for a viral receptor on a
potential host cell. For adenovirus, such manipulations can include
deletion of regions of the fiber, penton, or hexon, insertions of
various native or non-native ligands into portions of the coat
protein, and the like. Manipulation of the coat protein can broaden
the range of cells infected by a viral vector or enable targeting
of a viral vector to a specific cell type.
[0031] For example, in one embodiment, the adenoviral vector
comprises a chimeric coat protein (e.g., a fiber, hexon pIX, pIIIa,
or penton protein), which differs from the wild-type (i.e., native)
coat protein by the introduction of a normative amino acid
sequence, preferably at or near the carboxyl terminus. Preferably,
the normative amino acid sequence is inserted into or in place of
an internal coat protein sequence. One of ordinary skill in the art
will understand that the normative amino acid sequence can be
inserted within the internal coat protein sequence or at the end of
the internal coat protein sequence. The resultant chimeric viral
coat protein is able to direct entry into cells of the adenoviral,
vector comprising the coat protein that is more efficient than
entry into cells of a vector that is identical except for
comprising a wild-type adenoviral coat protein rather than the
chimeric adenoviral coat protein. Preferably, the chimeric
adenovirus coat protein binds a novel endogenous binding site
present on the cell surface that is not recognized, or is poorly
recognized, by a vector comprising a wild-type coat protein. One
direct result of this increased efficiency of entry is that the
adenovirus can bind to and enter numerous cell types which an
adenovirus comprising wild-type coat protein typically cannot enter
or can enter with only a low efficiency.
[0032] In another embodiment of the invention, the adenoviral
vector comprises a chimeric virus coat protein not selective for a
specific type of eukaryotic cell. The chimeric coat protein differs
from the wild-type coat protein by an insertion of a normative
amino acid sequence into or in place of an internal coat protein
sequence. In this embodiment, the chimeric adenovirus coat protein
efficiently binds to a broader range of eukaryotic cells than a
wild-type adenovirus coat, such as described in International
Patent Application WO 97/20051.
[0033] Specificity of binding of an adenovirus to a given cell can
also be adjusted by use of an adenovirus comprising a short-shafted
adenoviral fiber gene, as discussed in U.S. Pat. No. 5,962,311. Use
of an adenovirus comprising a short-shafted adenoviral fiber gene
reduces the level or efficiency of adenoviral fiber binding to its
cell-surface receptor and increases adenoviral penton base binding
to its cell-surface receptor, thereby increasing the specificity of
binding of the adenovirus to a given cell. Alternatively, use of an
adenovirus comprising a short-shafted fiber enables targeting of
the adenovirus to a desired cell-surface receptor by the
introduction of a normative amino acid sequence either into the
penton base or the fiber knob.
[0034] Of course, the ability of an adenoviral vector to recognize
a potential host cell can be modulated without genetic manipulation
of the coat protein. For instance, complexing an adenovirus with a
bispecific molecule comprising a penton base-binding domain and a
domain that selectively binds a particular cell surface binding
site enables one of ordinary skill in the art to target the vector
to a particular cell type.
[0035] Suitable modifications to an adenoviral vector are described
in U.S. Pat. Nos. 5,543,328, 5,559,099, 5,712,136, 5,731,190,
5,756,086, 5,770,442, 5,846,782, 5,871,727, 5,885,808, 5,922,315,
5,962,311, 5,965,541, 6,057,155, 6,127,525, 6,153,435, 6,329,190,
6,455,314, 6,465,253, 6,576,456, 6,649,407, 6,740,525; 6,951,755;
U.S. Patent Application Publications 2001/0047081 A1, 2002/0013286
A1, 2002/0151027 A1, 2003/0022355 A1, 2003/0099619 A1, 2003/0166286
A1, and 2004/0161848 A1; and International Patent Applications WO
95/02697, WO 95/16772, WO 95/34671, WO 96/07734, WO 96/22378, WO
96/26281, WO 97/20051, WO 98/07865, WO 98/07877, WO 98/40509, WO
98/54346, WO 00/15823, WO 01/58940, and WO 01/92549. Similarly, it
will be appreciated that numerous adenoviral vectors are available
commercially. Construction of adenoviral vectors is well understood
in the art. Adenoviral vectors can be constructed and/or purified
using methods known in the art (e.g., using complementing cell
lines, such as the 293 cell line, Per.C6 cell line, or 293-ORF6
cell line) and methods set forth, for example, in U.S. Pat. Nos.
5,965,358, 5,994,128, 6,033,908, 6,168,941, 6,329,200, 6,383,795,
6,440,728, 6,447,995, 6,475,757, 6,908,762, and 6,913,927; U.S.
Patent Application Publications 2002/0034735 A1 and 2004/0063203
A1; and International Patent Applications WO 98/53087, WO 98/56937,
WO 99/15686, WO 99/54441, WO 00/12765, WO 01/77304, and WO
02/29388, as well as the other references identified herein.
[0036] The adenoviral vector of the inventive method comprises an
adenoviral genome comprising a heterologous nucleic acid sequence.
A "heterologous nucleic acid sequence" is any nucleic acid sequence
that is not obtained from, derived from, or based upon a naturally
occurring nucleic acid sequence of the adenoviral vector. By
"naturally occurring" is meant that the nucleic acid sequence can
be found in nature and has not been synthetically modified. The
heterologous nucleic acid sequence also is not obtained from,
derived from, or based upon an adenoviral nucleic acid sequence.
For example, the heterologous nucleic acid sequence can be a viral,
bacterial, plant, or animal nucleic acid sequence. A sequence is
"obtained" from a source when it is isolated from that source. A
sequence is "derived" from a source when it is isolated from a
source but modified in any suitable manner (e.g., by deletion,
substitution (mutation), insertion, or other modification to the
sequence) so as not to disrupt the normal function of the source
gene. A sequence is "based upon" a source when the sequence is a
sequence more than about 70% homologous (preferably more than about
80% homologous, more preferably more than about 90% homologous, and
most preferably more than about 95% homologous) to the source but
obtained through synthetic procedures (e.g., polynucleotide
synthesis, directed evolution, etc.). Determining the degree of
homology, including the possibility for gaps, can be accomplished
using any suitable method (e.g., BLASTnr, provided by GenBank).
Notwithstanding the foregoing, the nucleic acid sequence that makes
up the heterologous nucleic acid sequence can be naturally found in
the adenoviral vector, but located at a normative position within
the adenoviral genome and/or operably linked to a normative
promoter.
[0037] The adenoviral vector comprises at least one heterologous
nucleic acid sequence as described herein, i.e., the adenoviral
vector can comprise one heterologous nucleic acid sequence as
described herein or more than one heterologous nucleic acid
sequence as described herein (i.e., two or more of the heterologous
nucleic acid sequences). The heterologous nucleic acid sequence
preferably encodes a protein (i.e., one or more nucleic acid
sequences encoding one or more proteins). An ordinarily skilled
artisan will appreciate that any type of nucleic acid sequence
(e.g., DNA, RNA, and cDNA) that can be inserted into an adenoviral
vector can be used in connection with the invention.
[0038] In the context of the invention, the heterologous nucleic
acid sequence can encode any suitable protein, but preferably
encodes a protein that is toxic to the cell. Desirably, the protein
is a bacterial protein, a viral protein, a plant protein, a
parasite protein, a fungi protein, an animal protein, or an
antibiotic. When the heterologous nucleic acid sequence encodes a
bacterial protein, the protein can be isolated or derived from any
suitable bacterium, including, but not limited to Actinomyces,
Anabaena, Bacillus, Bacteroides, Bdellovibrio, Caulobacter,
Chlamydia, Chlorobium, Chromatium, Clostridium, Cytophaga,
Deinococcus, Escherichia, Halobacterium, Heliobacter,
Hyphomicrobium, Methanobacterium, Micrococcus, Myobacterium,
Mycoplasma, Myxococcus, Neisseria, Nitrobacter, Oscillatoria,
Prochloron, Proteus, Pseudomonas, Phodospirillum, Rickettsia,
Salmonella, Shigella, Spirillum, Spirochaeta, Staphylococcus,
Streptococcus, Streptomyces, Sulfolobus, Thermoplasma,
Thiobacillus, and Treponema. Desirably, the heterologous nucleic
acid sequence encodes a toxin protein isolated or derived from
Bacillus anthracis (e.g., protective antigen, lethal factor, or
edema factor), Bordetella pertussis (e.g., adenylate cyclase toxin
or pertussis toxin), Vibrio cholerae (e.g., cholera enterotoxin),
Escherichia coli (e.g., ST toxin or LT toxin), Shigella dysenteriae
(e.g., shiga toxin), Clostridium perfringens (e.g., perfringens
enterotoxin), Clostridium botulinum (e.g., botulinum toxin),
Clostridium tetani (e.g., tetanus toxin), Corynebacterium
diphtheriae (e.g., diphtheria toxin), Pseudomonas aeruginosa (e.g.,
exotoxin A), Staphylococcus aureus (e.g., staphylococcus
enterotoxins, toxic shock syndrome toxin, or exfoliatin toxin), or
Streptococcus pyogenes (e.g., erythrogenic toxin).
[0039] The heterologous nucleic acid also can be encode a parasite
protein, such as, but not limited to, a parasite of the phylum
Sporozoa (also referred to as phylum Apicomplexa), Ciliophora,
Rhizopoda, or Zoomastigophora. Preferably, the parasite is of the
phylum Sporozoa and genus Plasmodium. The protein can be from any
suitable Plasmodium species, but preferably is from a Plasmodium
species that infects humans and causes malaria (e.g., Plasmodium
falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium
malariae). Suitable Plasmodium proteins include, for example,
circumsporozoite protein (CSP), sporozoite surface protein 2
(SSP2), liver-stage antigen 1 (LSA-1), Pf exported protein 1
(PfExp-1)/Py hepatocyte erythrocyte protein 17 (PyHEP17), Pf
Antigen 2, merozoite surface protein 1 (MSP-1), merozoite surface
protein 2 (MSP-2), erythrocyte binding antigen 175 (EBA-175),
ring-infected erythrocyte surface antigen (RESA), serine repeat
antigen (SERA), glycophorin binding protein (GBP-130), histidine
rich protein 2 (HRP-2), rhoptry-associated proteins 1 and 2 (RAP-1
and RAP-2), erythrocyte membrane protein 1 (PFEMP1), and apical
membrane antigen 1 (AMA-1).
[0040] When the heterologous nucleic acid sequence encodes a virus
protein, the protein can be isolated or derived from any suitable
virus including, but not limited to, a virus from any of the
following viral families: Arenaviridae, Arterivirus, Astroviridae,
Baculoviridae, Badnavirus, Barnaviridae, Birnaviridae,
Bromoviridae, Bunyaviridae, Caliciviridae, Capillovirus,
Carlavirus, Caulimovirus, Circoviridae, Closterovirus, Comoviridae,
Coronaviridae (e.g., Coronavirus, such as severe acute respiratory
syndrome (SARS) virus), Corticoviridae, Cystoviridae, Deltavirus,
Dianthovirus, Enamovirus, Filoviridae (e.g., Marburg virus and
Ebola virus (e.g., Zaire, Reston, Ivory Coast, or Sudan strain)),
Flaviviridae, (e.g., Hepatitis C virus, Dengue virus 1, Dengue
virus 2, Dengue virus 3, and Dengue virus 4), Hepadnaviridae (e.g.,
Hepatitis B virus), Herpesviridae (e.g., Human herpesvirus 1, 3, 4,
5, and 6, and Cytomegalovirus), Hypoviridae, Iridoviridae,
Leviviridae, Lipothrixviridae, Microviridae, Orthomyxoviridae
(e.g., Influenza virus A and B), Papovaviridae, Paramyxoviridae
(e.g., measles, mumps, and human respiratory syncytial virus),
Parvoviridae, Picornaviridae (e.g., poliovirus, rhinovirus,
hepatovirus, and aphthovirus), Poxyiridae (e.g., vaccinia virus),
Reoviridae (e.g., rotavirus), Retroviridae (e.g., lentivirus, such
as human immunodeficiency virus (HIV) 1 and HIV 2), Rhabdoviridae,
Totiviridae, Crimean-Congo haemorrhagic fever virus, Eastern Equine
Encephalitis virus, Hendra virus, Lassa fever virus, Monkeypox
virus, Nipah virus, Rift Valley fever virus, South American
Haemorrhagic Fever viruses, and Venezuelan Equine Encephalitis
virus.
[0041] Preferably, at least one protein of the inventive method is
a retroviral protein. The retroviral protein can be, for example,
an HIV antigen, such as all or part of the gag, env, or pol
proteins, or a fusion protein comprising any of the gag, env, or
pol proteins. Any clade of HIV is appropriate for protein
selection, including clades A, B, C, MN, and the like. Also
preferably, at least one protein encoded by the heterologous
nucleic acid sequence is a coronavirus protein, such as a SARS
virus protein. Suitable SARS virus proteins for the inventive
method include, for example, all or part of the E protein, the M
protein, and the spike protein of the SARS virus. In another
embodiment, at least one protein encoded by the heterologous
nucleic acid sequence is an aphthovirus protein, such as a
foot-and-mouth disease virus (FMDV) protein. Suitable FMDV proteins
include, for example, proteins 1A, 1B, 1C, and 1D, collectively
referred to as P1, which form the capsid proteins of the virus,
proteins 2A, 2B, and 2C (collectively referred to as the P2
protein), and proteins 3A, 3B, 3C, and 3D (collectively referred to
as the P3 protein). The FMDV protein also can be an empty virus
capsid of FMDV. An "empty virus capsid" contains only the portion
of the FMDV genome encoding the viral structural proteins and the
3C protein, which is required for capsid formation (see Mayr et
al., Virology, 263: 496-506 (1999)), and does not contain
infectious viral nucleic acid. Suitable viral proteins also include
all or part of Dengue protein M, Dengue protein E, Dengue D1NS1,
Dengue D1NS2, and Dengue D1NS3. The viral peptides specifically
recited herein are merely exemplary as any viral protein can be
used in the context of the invention.
[0042] When the heterologous nucleic acid sequence encodes a fungal
protein, the protein can be isolated or obtained from any of the
following genuses: Coccidioides, Candida, Cryptococcus,
Trichosporon, Acremonium, Cladophialophora, Pseudallescheria,
Rizopus, Scedosporium, Aspergillus, Aureobasidium, Bipolaris,
Fusarium, Phialophora, Blastomyces, Histoplasma, or Sporothrix.
[0043] When the heterologous nucleic acid sequence encodes a plant
protein, the plant protein can be any suitable protein naturally
produced by a plant, so long as it is toxic to animal cells (e.g.,
human cells). Suitable plant toxins include, but are not limited
to, lectins (e.g., ricin or abrin), alkaloids, glycosides,
oxalates, phenols, resins, volatile oils, and phototoxins (e.g.,
coumarins).
[0044] The heterologous nucleic acid sequence also can encode an
animal protein. In this regard, certain animal proteins are
inhibitory to adenovirus replication when such proteins are
produced in packaging cells. The heterologous nucleic acid sequence
can encode any suitable animal protein. Examples of suitable animal
proteins include, but are not limited to transforming growth factor
.beta. (TGF.beta.), or nitric oxide synthase (NOS).
[0045] In another embodiment, the heterologous nucleic acid
sequence can encode an antibiotic. The antibiotic can be isolated
from nature, synthetically generated, isolated from a genetically
engineered organism, and the like. The heterologous nucleic acid
sequence can encode any suitable antibiotic. Suitable antibiotics
include, but are not limited to, penicillin, ampicillin,
cephalosporin, griseofulvin, bacitracin, polymyxin B, amphotericin
B, erythromycin, neomycin, streptomycin, tetracycline, vancomycin,
gentamicin, rifamycin, and the like.
[0046] One of ordinary skill in the art will appreciate that many
of the aforementioned proteins, and portions thereof, can be
antigenic when produced in an animal (e.g., mammalian) cell. An
"antigen" is a molecule that triggers an immune response in a
mammal. An "immune response" can entail, for example, antibody
production and/or the activation of immune effector cells. Thus,
the heterologous nucleic acid sequence can encode an antigen which
comprises any subunit of any proteinaceous molecule, including a
protein or peptide of viral, bacterial, parasitic, fungal,
protozoan, prion, cellular, or extracellular origin, which ideally
provokes an immune response in mammal, preferably leading to
protective immunity. The heterologous nucleic acid sequence also
can encode a self antigen, i.e., an autologous protein which the
body reacts to as if it is a foreign invader.
[0047] Preferably, the nucleic acid is operably linked to (i.e.,
under the transcriptional control of) one or more promoter and/or
enhancer elements, for example, as part of a promoter-variable
expression cassette. Techniques for operably linking sequences
together are well known in the art. A "promoter" is a DNA sequence
that directs the binding of RNA polymerase and thereby promotes RNA
synthesis. A nucleic acid sequence is "operably linked" to a
promoter when the promoter is capable of directing transcription of
that nucleic acid sequence. A promoter can be native or non-native
to the nucleic acid sequence to which it is operably linked.
[0048] Any promoter (i.e., whether isolated from nature or produced
by recombinant DNA or synthetic techniques) can be used in
connection with the invention to provide for transcription of the
nucleic acid sequence. The promoter preferably is capable of
directing transcription in a eukaryotic (desirably mammalian) cell.
The functioning of the promoter can be altered by the presence of
one or more enhancers and/or silencers present on the vector.
"Enhancers" are cis-acting elements of DNA that stimulate or
inhibit transcription of adjacent genes. An enhancer that inhibits
transcription also is termed a "silencer." Enhancers differ from
DNA-binding sites for sequence-specific DNA binding proteins found
only in the promoter (which also are termed "promoter elements") in
that enhancers can function in either orientation, and over
distances of up to several kilobase pairs (kb), even from a
position downstream of a transcribed region.
[0049] Promoter regions can vary in length and sequence and can
further encompass one or more DNA binding sites for
sequence-specific DNA binding proteins and/or an enhancer or
silencer. Enhancers and/or silencers can similarly be present on a
nucleic acid sequence outside of the promoter per se. Desirably, a
cellular or viral enhancer, such as the cytomegalovirus (CMV)
immediate-early enhancer, is positioned in the proximity of the
promoter to enhance promoter activity. In addition, splice acceptor
and donor sites can be present on a nucleic acid sequence to
enhance transcription.
[0050] Any suitable promoter or enhancer sequence can be used in
the context of the invention. In this respect, the heterologous
nucleic acid sequence can be operably linked to a viral promoter.
Suitable viral promoters include, for instance, cytomegalovirus
(CMV) promoters, such as the CMV immediate-early promoter
(described in, for example, U.S. Pat. Nos. 5,168,062 and 5,385,839,
and GenBank accession number X17403), promoters derived from human
immunodeficiency virus (HIV), such as the HIV long terminal repeat
promoter, Rous sarcoma virus (RSV) promoters, such as the RSV long
terminal repeat, mouse mammary tumor virus (MMTV) promoters, HSV
promoters, such as the Lap2 promoter or the herpes thymidine kinase
promoter (Wagner et al., Proc. Natl. Acad. Sci., 78, 144-145
(1981)), promoters derived from SV40 or Epstein Barr virus, an
adeno-associated viral promoter, such as the p5 promoter, and the
like.
[0051] Alternatively, the heterologous nucleic acid sequence can be
operably linked to a cellular promoter, i.e., a promoter that
drives expression of a cellular protein. Preferred cellular
promoters for use in the invention will depend on the desired
expression profile to produce the antigen(s). In one aspect, the
cellular promoter is preferably a constitutive promoter that works
in a variety of cell types, such as immune cells. Suitable
constitutive promoters can drive expression of genes encoding
transcription factors, housekeeping genes, or structural genes
common to eukaryotic cells. For example, the Ying Yang 1 (YY1)
transcription factor (also referred to as NMP-1, NF-E1, and UCRBP)
is a ubiquitous nuclear transcription factor that is an intrinsic
component of the nuclear matrix (Guo et al., PNAS, 92, 10526-10530
(1995)). While the promoters described herein are considered as
constitutive promoters, it is understood in the art that
constitutive promoters can be upregulated. Promoter analysis shows
that the elements critical for basal transcription reside from -277
to +475 of the YY1 gene relative to the transcription start site
from the promoter, and include a TATA and CCAAT box. JEM-1 (also
known as HGMW and BLZF-1) also is a ubiquitous nuclear
transcription factor identified in normal and tumorous tissues
(Tong et al., Leukemia, 12 (11), 1733-1740 (1998), and Tong et al.,
Genomics, 69 (3), 380-390 (2000)). JEM-1 is involved in cellular
growth control and maturation, and can be upregulated by retinoic
acids. Sequences responsible for maximal activity of the JEM-1
promoter have been located at -432 to +101 of the JEM-1 gene
relative the transcription start site of the promoter. Unlike the
YY1 promoter, the JEM-1 promoter does not comprise a TATA box. The
ubiquitin promoter, specifically UbC, is a strong constitutively
active promoter functional in several species. The UbC promoter is
further characterized in Marinovic et al., J. Biol. Chem., 277
(19), 16673-16681 (2002).
[0052] In the inventive method, the heterologous nucleic acid
sequence is operably linked to a promoter and one or more operator
sequences of the tetracycline operon (tetO). The tetracycline
operon was originally identified in the Tn10 transposon, in which
it regulates the expression of tetracycline resistance genes (see,
e.g., Hillen et al. in Protein-Nucleic Acid Interaction, Topics in
Molecular and Structural Biology, Saenger et al., eds., Vol. 10,
143-162, Macmillan, London (1989)). The tetracycline operon, and
modified forms thereof, are used in the art to regulate gene
expression in recombinant DNA systems. In this regard, the
tetracycline regulation system consists of two components: operator
sequences (tetO) and a repressor protein (tetR). In the absence of
tetracycline, the tetR protein is able to bind to the tetO sites
and repress transcription of a gene operably linked to the tetO
sites. In the presence of tetracycline, however, a conformational
change in the tetR protein prevents it from binding to the operator
sequences, allowing transcription of operably linked genes to
occur. The tetracycline regulation system has been modified for use
in mammalian cells by the generation of a fusion protein combining
tetR with the transcriptional activation domain of the VP16 protein
of herpes simplex virus, which also is referred to as the tet
transactivator protein (tTa) (see, e.g., Gossen and Bujard, Proc.
Natl. Acad. Sci. USA, 89, 5547-5551 (1992), and Shockett and
Schatz, Proc. Natl. Acad. Sci. USA, 93, 5173-5176 (1996)).
[0053] The heterologous nucleic acid sequence can be operably
linked to any suitable tetO site and any suitable number of tetO
sites, so long as expression of the heterologous nucleic acid
sequence is inhibited in the presence of tetR. In a preferred
embodiment of the invention, the heterologous nucleic acid sequence
is operably linked to one or more tetO sites, each of which
comprises the nucleotide sequence
AGCTCTCCCTATCAGTGATAGAGATCTCCCTATCAGTGATAGAGATCGTCGACGA GCT (SEQ ID
NO: 1). The heterologous nucleic acid sequence preferably is
operably linked to at least one (e.g., 1, 2, 3, 4, 5, 6, or more)
tetO sequence, but more preferably is operably linked to at least
two (e.g., 2, 3, 4, 5, 6, or more) tetO sequences.
[0054] The one or more tetO sequences can be located in any
suitable position with respect to the heterologous nucleic acid
sequence and the promoter. In this regard, the tetO sequences can
be located upstream of both the promoter and the heterologous
nucleic acid sequence. Alternatively, the tetO sequences can be
located between the promoter and the heterologous nucleic acid
sequence. In another embodiment, the one or more tetO sequences can
be located downstream of both the promoter and the heterologous
nucleic acid sequence. In addition, the one or more tetO sequences
need not be positioned in tandem. For example, one tetO sequence
can be located upstream of the promoter, while a second tetO
sequence can be located downstream of the promoter and upstream of
the heterologous nucleic acid sequence.
[0055] Operable linkage of a heterologous nucleic acid sequence to
a promoter and tetO sequences is within the skill of the art, and
can be accomplished using routine recombinant DNA techniques, such
as those described in, for example, Sambrook et al., Molecular
Cloning, a Laboratory Manual, 2d edition, Cold Spring Harbor Press,
Cold Spring Harbor, N.Y. (1989), and Ausubel et al., Current
Protocols in Molecular Biology, Greene Publishing Associates and
John Wiley & Sons, New York, N.Y. (1994).
[0056] To optimize protein production, preferably the heterologous
nucleic acid sequence further comprises a polyadenylation site
following the coding sequence of the heterologous nucleic acid
sequence. Any suitable polyadenylation sequence can be used,
including a synthetic optimized sequence, as well as the
polyadenylation sequence of BGH (Bovine Growth Hormone), polyoma
virus, TK (Thymidine Kinase), EBV (Epstein Barr Virus), and the
papillomaviruses, including human papillomaviruses and BPV (Bovine
Papilloma Virus). A preferred polyadenylation sequence is the SV40
(Human Sarcoma Virus-40) polyadenylation sequence. Also, preferably
all the proper transcription signals (and translation signals,
where appropriate) are correctly arranged such that the nucleic
acid sequence is properly expressed in the cells into which it is
introduced. If desired, the nucleic acid sequence also can
incorporate splice sites (i.e., splice acceptor and splice donor
sites) to facilitate mRNA production.
[0057] The construction of adenoviral vectors is well understood in
the art. Adenoviral vectors can be constructed and/or purified
using the methods set forth, for example, in U.S. Pat. Nos.
5,965,358, 6,168,941, 6,329,200, 6,383,795, 6,440,728, 6,447,995,
6,475,757, 6,573,092, and 6,586,226, and U.S. Patent Application
Publication Nos. 2003/0170899 A1, 2003/0203469 A1, and 2003/0203480
A1, and International Patent Application Publications WO 98/53087,
WO 98/56937, WO 99/15686, WO 99/54441, WO 00/12765, WO 01/77304, WO
02/29388, WO 02/31169, and WO 03/39459 as well as the other
references identified herein. Non-group C adenoviral vectors,
including adenoviral serotype 35 vectors, can be produced using the
methods set forth in, for example, U.S. Pat. Nos. 5,837,511 and
5,849,561, and International Patent Application Publications WO
97/12986 and WO 98/53087. Moreover, numerous adenoviral vectors are
available commercially.
[0058] The inventive method further comprises providing a cell
having a cellular genome comprising a nucleic acid sequence
encoding a tetracycline operon repressor protein (tetR). The cell
can be any suitable cell which can propagate adenoviral vectors
when infected with such vectors or with nucleic acid sequences
encoding the adenoviral genome. In this regard, the cell desirably
comprises a genome that can incorporate and preferably retain a
nucleic acid encoding an adenoviral gene product that complements
in trans for a deficiency in one or more regions of an adenoviral
genome. Most preferably, the cell can propagate a suitable
replication-deficient adenoviral vector upon infection with an
appropriate replication-deficient adenoviral vector or transfection
with an appropriate replication-deficient viral genome.
[0059] Particularly desirable cell types are those that support
high levels of adenovirus propagation. The cell preferably produces
at least about 10,000 viral particles per cell and/or at least
about 3,000 focus forming units (FFU) per cell. More preferably,
the cell produces at least about 100,000 viral particles per cell
and/or at least about 5,000 FFU per cell. Most preferably, the cell
produces at least about 200,000 viral particles per cell and/or at
least about 7,000 FFU per cell.
[0060] Preferably, the cell is, or is derived from, an anchorage
dependent cell, but which has the capacity to grow in suspension
cultures. In one embodiment, the cell can be a primary cell. By
"primary cell" is meant that the cell does not replicate
indefinitely in culture. Examples of suitable primary cells
include, but are not limited to, human embryonic kidney (HEK)
cells, human retinal cells, and human embryonic retinal (HER)
cells. In another embodiment, the cell can be a transformed cell.
The cell is "transformed" in that the cell has the ability to
replicate indefinitely in culture. Examples of suitable transformed
cells include renal carcinoma cells, CHO cells, KB cells, HEK-293
cells, SW-13 cells, MCF7 cells, HeLa cells, Vero cells, neural
cells (e.g., BE(2)-M17 cells and SK-N-MC cells), and lung carcinoma
cells. Complementing cell lines for producing the adenoviral vector
include, but are not limited to, 293 cells (described in, e.g.,
Graham et al., J. Gen. Virol., 36, 59-72 (1977)), PER.C6 cells
(described in, e.g., International Patent Application WO 97/00326,
and U.S. Pat. Nos. 5,994,128 and 6,033,908), and 293-ORF6 cells
(described in, e.g., International Patent Application WO 95/34671
and Brough et al., J. Virol., 71, 9206-9213 (1997)). Additional
complementing cells are described in, for example, U.S. Pat. Nos.
6,677,156 and 6,682,929, and International Patent Application
Publication WO 03/20879.
[0061] The cell comprises a cellular genome comprising a nucleic
acid sequence encoding a tetracycline operon repressor protein
(tetR). Like the tetO sequence, the tetR protein was originally
identified in the Tn10 transposon as part of the tetracycline
operon (see Hillen et al., supra). The tetR protein preferably
comprises the amino acid sequence of SEQ ID NO: 2 (GenBank
Accession No. J01830, GI No. 154846).
[0062] While it is preferred that the nucleic acid sequence
encoding the tetR protein encodes a wild-type tetR protein (such as
is set forth in SEQ ID NO: 2), the nucleic acid sequence
alternatively can encode any suitable variant of the tetR protein.
A variant of the tetR protein retains the ability to bind to tetO
sequences and repress transcription of a nucleic acid sequence
operably linked thereto. A variant tetR protein preferably is
produced by introducing one or more mutations (e.g., point
mutations, deletions, insertions, etc.) into the nucleic acid
sequence encoding a wild type tetR protein. Such mutations are
introduced in the nucleic acid sequence to effect one or more amino
acid substitutions in an encoded tetR protein. Thus, where
mutations are introduced in the nucleic acid sequence encoding the
tetR protein, such mutations desirably will effect a substitution
in the encoded tetR protein whereby codons encoding
positively-charged residues (H, K, and R) are substituted with
codons encoding positively-charged residues, codons encoding
negatively-charged residues (D and E) are substituted with codons
encoding negatively-charged residues, codons encoding neutral polar
residues (C, G, N, Q, S, T, and Y) are substituted with codons
encoding neutral polar residues, and codons encoding neutral
non-polar residues (A, F, I, L, M, P, V, and W) are substituted
with codons encoding neutral non-polar residues. In addition, the
nucleic acid sequence can encode a homolog of a tetR protein. A
homolog of a tetR protein, whether wild-type or mutant, can be any
peptide, polypeptide, or portion thereof, that is more than about
70% identical (preferably more than about 80% identical, more
preferably more than about 90% identical, and most preferably more
than about 95% identical) to the tetR protein at the amino acid
level. The degree of amino acid identity can be determined using
any method known in the art, such as the BLAST sequence database.
Furthermore, a homolog of the tetR protein can be any peptide,
polypeptide, or portion thereof, which hybridizes to the tetR
protein under at least moderate, preferably high, stringency
conditions. Exemplary moderate stringency conditions include
overnight incubation at 37.degree. C. in a solution comprising 20%
formamide, 5.times.SSC (150 mM NaCl, 15 mM trisodium citrate), 50
mM sodium phosphate (pH 7.6), 5.times.Denhardt's solution, 10%
dextran sulfate, and 20 mg/ml denatured sheared salmon sperm DNA,
followed by washing the filters in 1.times.SSC at about
37-50.degree. C., or substantially similar conditions, e.g., the
moderately stringent conditions described in Sambrook et al.,
supra. High stringency conditions are conditions that use, for
example (1) low ionic strength and high temperature for washing,
such as 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium
dodecyl sulfate (SDS) at 50.degree. C., (2) employ a denaturing
agent during hybridization, such as formamide, for example, 50%
(v/v) formamide with 0.1% bovine serum albumin (BSA)/0.1%
Ficoll/0.1% polyvinylpyrrolidone (PVP)/50 mM sodium phosphate
buffer at pH 6.5 with 750 mM sodium chloride and 75 mM sodium
citrate at 42.degree. C., or (3) employ 50% formamide, 5.times.SSC
(0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH
6.8), 0.1% sodium pyrophosphate, 5.times.Denhardt's solution,
sonicated salmon sperm DNA (50 .mu.g/ml), 0.1% SDS, and 10% dextran
sulfate at 42.degree. C., with washes at (i) 42.degree. C. in
0.2.times.SSC, (ii) 55.degree. C. in 50% formamide, and (iii)
55.degree. C. in 0.1.times.SSC (preferably in combination with
EDTA). Additional details and an explanation of stringency of
hybridization reactions are provided in, e.g., Ausubel et al.,
supra.
[0063] Replication-deficient adenoviral vectors are typically
produced in complementing cell lines that provide gene functions
not present in the replication-deficient adenoviral vectors, but
required for viral propagation, at appropriate levels in order to
generate high titers of viral vector stock. Thus, in addition to
the nucleic acid sequence encoding a tetR protein, the cell
preferably comprises, integrated into the cellular genome,
adenoviral nucleic acid sequences which encode gene functions
required for adenoviral propagation. A preferred cell complements
for at least one and preferably all replication-essential gene
functions not present in a replication-deficient adenovirus. The
cell can complement for a deficiency in at least one
replication-essential gene function encoded by the early regions,
late regions, viral packaging regions, virus-associated RNA
regions, or combinations thereof, including all adenoviral
functions (e.g., to enable propagation of adenoviral amplicons).
Most preferably, the cell complements for a deficiency in at least
one replication-essential gene function (e.g., two or more
replication-essential gene functions) of the E1 region of the
adenoviral genome, particularly a deficiency in a
replication-essential gene function of each of the E1A and E1B
regions. In addition, the cell can complement for a deficiency in
at least one replication-essential gene function of the E2
(particularly as concerns the adenoviral DNA polymerase and
terminal protein) and/or E4 regions of the adenoviral genome.
[0064] Desirably, a cell that complements for a deficiency in the
E4 region comprises the E4-ORF6 gene sequence and produces the
E4-ORF6 protein. Such a cell desirably comprises at least ORF6 and
no other ORF of the E4 region of the adenoviral genome. The ORF-6
of the E4 region of the adenoviral genome preferably is an ORF-6 of
the E4 region of a human adenoviral genome, such as a serotype 5 or
serotype 2 adenoviral genome. In addition, the ORF-6 of the E4
region of the adenoviral genome can be operably linked to any
suitable promoter, but preferably is operably linked to an
inducible promoter. Any suitable inducible promoter may be used to
regulate the ORF-6 of the E4 region of the adenoviral genome, and
suitable inducible promoters are known in the art. In a preferred
embodiment of the invention, the inducible promoter is a sheep
metallothionine promoter.
[0065] The cell preferably is further characterized in that it
contains the complementing genes in a non-overlapping fashion with
the adenoviral vector, which minimizes, and practically eliminates,
the possibility of the vector genome recombining with the cellular
DNA. Accordingly, the presence of replication competent
adenoviruses (RCA) is minimized if not avoided in the vector stock,
which, therefore, is suitable for certain therapeutic purposes,
especially vaccination purposes. The lack of RCA in the vector
stock avoids the replication of the adenoviral vector in
non-complementing cells. Construction of such a complementing cell
involves standard molecular biology and cell culture techniques,
such as those described by Sambrook et al., supra, and Ausubel et
al., supra).
[0066] In some instances, the cellular genome need not comprise
nucleic acid sequences, the gene products of which complement for
all of the replication-essential deficiencies of a
replication-deficient adenoviral vector. One or more
replication-essential gene functions lacking in a
replication-deficient adenoviral vector can be supplied by a helper
virus, e.g., an adenoviral vector that supplies in trans one or
more essential gene functions required for replication of the
desired adenoviral vector. Helper virus is often engineered to
prevent packaging of infectious helper virus. For example, one or
more replication-essential gene functions of the E1 region of the
adenoviral genome can be provided by the complementing cell, while
one or more replication-essential gene functions of the E4 region
of the adenoviral genome can be provided by a helper virus.
[0067] In accordance with the inventive method, the adenoviral
vector contacts the cell so as to transfect the cell with the
adenoviral vector, such that (a) the nucleic acid sequence encoding
tetR is expressed to produce tetR, (b) expression of the
heterologous nucleic acid sequence is inhibited in the presence of
tetR, and (c) the adenoviral vector is propagated. The cell can be
contacted with the adenoviral vector using any suitable method
known in the art. Preferably, the cell is transfected with the
adenoviral vector in vitro using standard techniques (e.g., calcium
phosphate precipitated transfection).
[0068] Upon uptake of the adenoviral vector by the cell, the tetR
protein produced by the cell desirably binds to the one or more
tetO sequences operably linked to the heterologous nucleic acid
sequence of the adenoviral vector. As discussed above, tetR binding
to tetO sequences prevents the transcriptional machinery from
accessing the promoter operably linked to the heterologous nucleic
acid sequence, thereby inhibiting expression of the heterologous
nucleic acid sequence. The expression of the heterologous nucleic
acid sequence is "inhibited" when the level of expression
(typically and preferably transcription) of the heterologous
nucleic acid sequence in the presence of tetR is at most about 80%
(e.g., no more than about 80%, about 70%, or about 60%) the level
of expression of the heterologous nucleic acid sequence in the
absence of tetR. Preferably, the level of expression of the
heterologous nucleic acid sequence in the presence of tetR is at
most about 50% (e.g., no more than about 50%, about 40%, or about
30%) the level of expression of the heterologous nucleic acid
sequence in the absence of tetR. More preferably, the level of
expression of the heterologous nucleic acid sequence in the
presence of tetR is at most about 20% (e.g., no more than about
20%, about 10%, about or about 5%) the level of expression of the
heterologous nucleic acid sequence in the absence of tetR. Ideally,
expression of the heterologous nucleic acid sequence is completely
inhibited in the presence of tetR.
[0069] One of ordinary skill in the art will appreciate that
expression of the nucleic acid sequence encoding the tetR protein
in the cell increases the yield of adenoviral vectors encoding the
heterologous nucleic acid sequence per cell when compared to the
yield of adenoviral vectors per cell when the nucleic acid sequence
encoding tetR is not expressed in the cell. Expression of the tetR
protein preferably increases the yield of adenoviral vector at
least about 5-fold, and more preferably increases the yield of
adenoviral vector at least about 20-fold, as compared to the yield
of adenoviral vectors when the cell does not express the tetR
protein.
[0070] The invention further provides a system comprising a cell
comprising a cellular genome comprising a nucleic acid sequence
encoding a tetracycline operon repressor protein (tetR), which can
be expressed to produce tetR, and an adenoviral vector. The
adenoviral vector has an adenoviral genome comprising a
heterologous nucleic acid sequence encoding a protein that is toxic
to the cell, wherein the heterologous nucleic acid sequence is
operably linked to a promoter and one or more tetracycline operon
operator sequences (tetO), and wherein the adenoviral vector can
transfect the cell and be propagated in the cell. Descriptions of
the adenoviral vector, the tetO sequences, the cell, and the tetR
protein set forth above in connection with other embodiments of the
invention also are applicable to those same aspects of the
aforesaid system.
[0071] The following examples further illustrate the invention but,
of course, should not be construed as in any way limiting its
scope.
EXAMPLE 1
[0072] This example demonstrates a method of inhibiting gene
expression from an adenoviral vector according to the inventive
method.
[0073] An oligonucleotide containing two copies of the tet operator
(5'-AGCTCTCCCTATCAGTGATAGAGATCTCCCTATCAGTGATAGAGATCGTCGACGA GCT-3')
(SEQ ID NO: 1) was self-annealed, digested with SacI, and inserted
at the SacI site between the TATA box and transcription start site
of the CMV enhancer/promoter (GenBank X17403, nucleotides 174,314
to 173,566). An artificial untranslated sequence (UTR) of 144 base
pairs and 3' splice site sequences were inserted downstream of the
CMV sequences, followed by a nucleic acid sequence encoding green
fluorescent protein (GFP) and a simian virus-40 (SV40)
polyadenylation signal. The resulting CMV-tetO expression cassette
was transferred to a shuttle plasmid containing adenovirus type 5
nucleotides 1-355 and 3333-5793 or 3511-5793 flanking the
expression cassette and a restriction site for linearization.
[0074] Adenoviral vector genomes were constructed using the AdFast
method (see U.S. Pat. No. 6,329,200). Briefly, E. coli strain BJDE3
was transfected with 100 ng of shuttle plasmid containing the
CMV-tetO expression cassette and 100 ng of a GV.11 base plasmid.
The desired recombinant plasmids, containing deletions in the E1,
E3, and E4 regions of the adenoviral genome and the expression
cassette were identified by restriction digestion of DNA from
individual bacterial colonies. The plasmids were further purified
by transformation of recombination negative DH5a E. coli and
single-colony isolation by standard microbiological methods.
Isolation of a single genetic clone of the final vector genome was
achieved by two sequential colony-growth steps in bacteria. The
adenoviral vector plasmid structures were confirmed by restriction
digestion analysis and DNA sequencing.
[0075] Cell populations of 293 and 293-ORF6 (293 cells that express
the Ad5 34 kDa E4 ORF6 protein (Brough et al., J. Virol., 70,
6497-6501 (1996)) carrying the episomal plasmid
pREPrsv(Koz-tetR)BghpA (293TetR and 293-ORF6TetR, respectively)
were generated by transfection of 2 .mu.g of circular plasmid and
addition of hygromycin to 150 .mu.g/ml in the cell culture medium.
The transfected cell populations were maintained under hygromycin
selection. The expression of tetR protein was confirmed by Western
blot analysis of 293TetR and 293-ORF6TetR extracts, which
demonstrated that the cell lines that maintained the tetR episome
were expressing tetR protein. To generate the stable transfectant
cell line, 293-ORF6NT, 293-ORF6 cells were transfected with 2 .mu.g
of HpaI-linearized pRSVTetR.hyg plasmid. After 24 hours the cells
were split to ten 10 cm dishes and incubated in 250 .mu.g/ml
hygromycin.
[0076] To demonstrate functional repressor activity, 293TetR cells
were transduced with the above-described E1-, E3-, E4-deleted,
adenoviral vectors (Rasmussen et al., Cancer Gene Ther., 9, 951-957
(2002)) comprising a nucleic acid sequence encoding GFP expressed
under the control of either a CMV promoter (Adf.11D) or the
CMV-tetO promoter (AdtetO.f.11D). The fluorescence intensity of
293TetR cells transduced with AdtetO.f.11D was much lower than that
of cells transduced by Adf.11D. The addition of 2 .mu.g/ml
doxycycline (dox) to cultures of 293TetR cells transduced by the
AdtetO.f.11D adenoviral vector resulted in fluorescent intensity
similar to that of 293TetR cells transduced by Adf.11D.
[0077] To determine whether the lower protein levels observed with
the tetR/tetO system were due to reduced levels of transcription,
the relative steady-state levels of GFP mRNA were determined by
Northern blot analysis of cells productively infected with 1 or 10
focus forming units (FFU) per cell of AdtetO.f.11D. Steady-state
GFP mRNA was reduced early (6 hours post-infection or h.p.i.) and
late (24 h.p.i.) in 293-ORF6TetR cells compared to 293-ORF6. Thus,
the lower level of protein products during virus replication was
due to repression of transcription. Moreover, binding of tetR
protein to adenovirus DNA did not affect virus propagation or
growth since there was no difference between marker gene vectors
with and without the tetR/tetO system.
[0078] The results of this example demonstrate that gene expression
from an adenoviral vector comprising tetO sequences can be
inhibited in cells expressing a functional tetR protein.
EXAMPLE 2
[0079] This example demonstrates a method of inhibiting gene
expression from an adenoviral vector according to the inventive
method.
[0080] 293 cells expressing tetR (293TetR) and lacking tetR (293BB)
were generated as described in Example 1. Cells were infected with
E1-deleted adenoviral vectors that expressed secreted alkaline
phosphatase (SEAP) from constitutive (AdSeap) and regulatable
(AdTetO.Seap) expression cassettes. The level of SEAP activity in
the culture medium was determined (Phospha-Light.TM. Kit, Applied
Biosystems, Foster City, Calif.) at three early phase time points:
8, 10, and 12 hours post-infection (h.p.i.) and at one time point
after significant DNA replication (24 h.p.i.). The level of SEAP
activity was specifically reduced in the 293TetR cells infected
with AdtetO.Seap. SEAP activity was not reduced in 293BB cells
infected with AdtetO.Seap, as compared to 293BB cells infected with
AdSeap. The level of SEAP activity was reduced by the tetO
vector-tetR cell combination more than 10-fold at the early time
points, although the reduction in activity decreased to
approximately 3-fold by 24 h.p.i. There was no effect of the
repressor on SEAP expression from the adenoviral vector that did
not contain tetO sequences (i.e., AdSeap+293TetR compared to
AdSeap+293BB).
[0081] Similar experiments were performed using E1-, E3-,
E4-deleted adenoviral vectors. In particular, 293-ORF6 cells and
293-ORF6TetR cells (see Example 1) were infected with SEAP-encoding
adenoviral vectors AdS.11D and AdTetO.S.11D. AdS.11D and
AdTetO.S.11D expressed comparable amounts of SEAP in 293-ORF6
cells. In 293-ORF6TetR cells, the expression of SEAP by
AdTetO.S.11D was significantly reduced as compared to SEAP
expression by AdTetO.S.11D in 293-ORF6 cells. The greatest
differences in SEAP activity between AdTetO.S.11D infected 293-ORF6
and 293-ORF6TetR cells occurred at the early phase time points of 6
and 8 h.p.i. (>10-fold). SEAP activity was reduced about 7 fold
at 12 h.p.i., and about 3-fold at 24 h.p.i. The addition of 2
.mu.g/ml doxycycline (dox) to cultures of 293-ORF6TetR cells at the
time of infection with AdTetO.S.11D resulted in the expression of
SEAP.
[0082] The results of this example demonstrate that gene expression
from an adenoviral vector comprising tetO sequences can be
inhibited in cells expressing a functional tetR protein early in
the virus growth cycle. The results of this example also suggest
that inhibition of gene expression is affected by the number of
vector genomes present within the cell.
EXAMPLE 3
[0083] This example demonstrates a method of propagating an
adenoviral vector comprising a nucleic acid sequence encoding a
toxic protein in accordance with the inventive method.
[0084] Seven adenoviral vectors comprising an expression cassette
under the control of a CMV promoter inserted into a deleted E1
region could not be propagated to form a stock of viable adenoviral
vectors using standard 293 or 293-ORF6 cells. The transgenes
encoded by the adenoviral vectors included human transforming
growth factor beta-1 (TGF.beta.) (Wettergreen et al., Eur. J. Oral
Sci., 109, 415-421 (2001), two peptide antibiotics, two viral
envelope glycoproteins, and two malaria parasite proteins. In
contrast, adenoviral vectors encoding each of these genes could be
propagated using the tetR/tetO system described herein. To
illustrate, an E1-deleted adenoviral vector comprising the CMV-tetO
expression cassette was constructed in accordance with the
description herein (see, e.g., Example 1). The adenoviral vector
encoded an activated form of TGF.beta. known to have a three to
five-fold higher biological activity than wild-type TGF.beta.
(Brunner et al., J. Biol. Chem., 264, 13660-13664 (1989))
(AdtetO.TGF.beta.). To determine the effect of high level TGF.beta.
expression on adenoviral vector propagation, 293 and 293TetR cells
were infected with AdtetO.TGF.beta.. At 12 hours post-infection,
approximately 1.7-fold more TGF.beta. was detected in the culture
medium of 293 cells as compared to 293TetR cells. However, by 24
h.p.i., significantly more TGF.beta. had accumulated in the culture
medium of 293TetR cells (t-test, p=0.01). Despite the high level of
TGF.beta. protein accumulation, there was a 3-fold higher yield of
infectious viral particles from 293TetR cells at 24 hours
post-infection.
[0085] The results of this example demonstrate that an adenoviral
vector encoding a toxic protein (such as TGF.beta.) can be
propagated in accordance with the inventive method. The results of
this example also suggest that adenoviral propagation is less
refractory to toxic protein, specifically TGF.beta., inhibition
late in infection.
EXAMPLE 4
[0086] This example demonstrates a method of propagating an
adenoviral vector comprising a nucleic acid sequence encoding a
toxic protein in accordance with the inventive method.
[0087] An adenoviral vector expressing high levels of a modified
HIV-1 envelope gene, gp140 (Yang et al., J. Virol., 78, 4029-4036
(2004)) could not be efficiently propagated. Four attempts to
propagate the adenoviral vector failed before the fifth attempt was
successful. The yield of virus progeny in the fifth attempt was
10-fold lower than expected.
[0088] Three different approaches were attempted to overcome the
inhibition to vector generation and growth. First, the gp140
nucleic acid sequence was altered to remove potential inhibitory
areas of the gp140 envelope protein. Deletion of protein coding
regions of gp140 to generate gp140dV12 (Yang et al., supra)
resulted in a high level of gp140 gene expression. The second
approach entailed deleting the introns from the CMV expression
cassette (hCMV.DELTA.) to decrease the level of gp140 expression.
This modification resulted in a 10-fold decrease in gp140
expression. While both of these approaches were successful at
restoring efficient generation of the adenoviral vectors and
improved adenovirus production yield, the modifications to the
gp140 coding sequence and expression thereof were significant. In
contrast, the third approach, which entailed inhibiting gp140
expression using the tetR/tetO tet system discussed above, resulted
in efficient viral propagation and high virus yield without
altering the gene product or reducing the potency of the adenoviral
vector. A summary of the results is set forth in the following
table.
TABLE-US-00001 Relative Viral Relative Viral Propagation
Yield.sup.a Expression Success/ (# of Adenoviral Vector Cell Line
(%) Attempts preparations) hCMV.Luciferase 293-ORF6 100 1/1 100%
(6) hCMV.gp140 293-ORF6 100 1/5 6% (3) hCMV.gp140dV12 293-ORF6 100
1/1 88% (3) hCMVA.gp140 293-ORF6 10 3/3 84% (5) hCMVtetO.gp140
293-ORF6 100 0/1 n.a. hCMVtetO.gp140 293-ORF6TetR Repressed 1/1
100% (4) .sup.aRelative to the hCMV.Luciferase adenoviral
vector
[0089] The results of this example demonstrate that an adenoviral
vector encoding a toxic protein (such as gp140) can be propagated
in accordance with the inventive method.
EXAMPLE 5
[0090] This example demonstrates a method of propagating an
adenoviral vector comprising a nucleic acid sequence encoding a
toxic protein in accordance with the inventive method.
[0091] An E1-deleted adenoviral vector encoding human inducible
nitric oxide synthase (iNOS) was grown to high titer only if an
iNOS inhibitor was included in the culture medium, demonstrating
the inhibitory effect of iNOS overexpression on adenoviral vector
replication. Even with the use of inhibitors, however, the iNOS
adenoviral vector was propagated at a relatively low titer
(10.sup.9 particle forming units (PFU)/mL) (Shears et al., J. Am.
Coll. Surg., 187, 295-306 (1998)). The preparations of adenoviral
vectors containing a CMV-iNOS expression cassette were rapidly
overtaken by replication competent adenovirus (RCA) and mutated
vectors containing deletions of the iNOS expression cassette,
implying a selective pressure against the expression of iNOS.
[0092] To prevent RCA formation and alleviate the negative effects
of iNOS overexpression, an E1-, E3-, and E4-deleted adenoviral
vector containing a CMV-TetO-iNOS expression cassette was
constructed in accordance with the description herein. In addition,
a PCR assay was developed to assess the integrity of the expression
cassette. Two AdFAST vector plasmids with identical CMV-tetO-iNOS
expression cassettes were generated. The two adenoviral vectors
produced via the AdFAST method differed only in the fiber protein,
expressing either a wild-type fiber (AdtetO.hiNOS.11D) or a fiber
containing a seven amino acid C-terminal addition
(AdtetO.hiNOS.F(pK7).11D (Wickham et al., J. Virol., 71, 8221-8229
(1997)).
[0093] 293-ORF6 and 293-ORF6TetR cells were transfected with each
vector, and lysates were passaged in parallel until cytopathic
effect (c.p.e.) on the cells was observed. The iNOS adenoviral
vectors propagated on 293-ORF6TetR cells achieved sufficient titer
in two passages to generate greater than 50% c.p.e. of
1.times.10.sup.6 cells. Subsequent generation of cesium chloride
purified stocks yielded titers averaging 2.7.times.10.sup.11 FFU/mL
with an average particle:FFU ratio of 8. Transgene expression and
activity of iNOS was confirmed by quantitation of total nitric
oxide in transduced cell supernatants (R & D Systems,
Minneapolis, Minn.). In comparison, growth of the vectors on
293-ORF6 cells was much slower. Although the adenoviral genome of
AdtetO.iNOS.F(pK7).11D was detected by a PCR assay throughout the
virus passages on 293-ORF6 cells, the vector did not achieve
sufficient titer to induce c.p.e. on the cells even after seven
passages.
[0094] Infected cell lysates of equal vector passage number were
assayed for rearrangements of the expression cassette by PCR
analysis. The expected full length amplification product was
detected with all adenoviral vectors, and there were no unexpected
amplicons detectable in the adenoviral vector preparations
performed on 293-ORF6TetR cells. However, production and
propagation of the iNOS adenoviral vectors on 293-ORF6 cells
yielded unexpected amplicons smaller than the full length product
(i.e., approximately 2.1 kb in AdtetO.iNOS.11D and approximately
1.1 kb in AdtetO.iNOS.F(pK7).11D). The 2.1 kb and 1.1 kb PCR
products were purified from the agarose gel and sequenced. The 2.1
kb amplicon contained a 2.9 kb deletion of the 3-prime end of the
expression cassette consisting of 80% of the iNOS ORF and the
entire SV40 polyadenylation site. Similarly, the 1.1 kb amplicon
contained a 3.8 kb deletion of the 5-prime end of the expression
cassette consisting of the CMV promoter, leaving only 232 bases of
the CMV enhancer, and the entire iNOS ORF.
[0095] In addition, an E1-, E3-deleted CMV-tetO-iNOS adenoviral
vector was constructed by the AdFAST method, propagated on 293TetR
cells (Wang et al., Mol. Ther., 7, 597-603 (2003)), and was
demonstrated to be free of RCA (approximately 10.sup.10 pu tested),
and no E1 region deletions were detected by PCR. Thus, as a result
of transcriptional repression, the tetR/tetO system was effective
in preventing the overgrowth of cultures by adenovectors with
non-functional expression cassettes.
[0096] The results of this example demonstrate that an adenoviral
vector encoding a toxic protein (such as iNOS) can be propagated in
accordance with the inventive method.
[0097] All references, including publications, patent applications,
and patents, cited herein are hereby incorporated by reference to
the same extent as if each reference were individually and
specifically indicated to be incorporated by reference and were set
forth in its entirety herein.
[0098] The use of the terms "a" and "an" and "the" and similar
referents in the context of describing the invention (especially in
the context of the following claims) are to be construed to cover
both the singular and the plural, unless otherwise indicated herein
or clearly contradicted by context. The terms "comprising,"
"having," "including," and "containing" are to be construed as
open-ended terms (i.e., meaning "including, but not limited to,")
unless otherwise noted. Recitation of ranges of values herein are
merely intended to serve as a shorthand method of referring
individually to each separate value falling within the range,
unless otherwise indicated herein, and each separate value is
incorporated into the specification as if it were individually
recited herein. All methods described herein can be performed in
any suitable order unless otherwise indicated herein or otherwise
clearly contradicted by context. The use of any and all examples,
or exemplary language (e.g., "such as") provided herein, is
intended merely to better illuminate the invention and does not
pose a limitation on the scope of the invention unless otherwise
claimed. No language in the specification should be construed as
indicating any non-claimed element as essential to the practice of
the invention.
[0099] Preferred embodiments of this invention are described
herein, including the best mode known to the inventors for carrying
out the invention. Variations of those preferred embodiments may
become apparent to those of ordinary skill in the art upon reading
the foregoing description. The inventors expect skilled artisans to
employ such variations as appropriate, and the inventors intend for
the invention to be practiced otherwise than as specifically
described herein. Accordingly, this invention includes all
modifications and equivalents of the subject matter recited in the
claims appended hereto as permitted by applicable law. Moreover,
any combination of the above-described elements in all possible
variations thereof is encompassed by the invention unless otherwise
indicated herein or otherwise clearly contradicted by context.
Sequence CWU 1
1
2158DNAEscherichia coli 1agctctccct atcagtgata gagatctccc
tatcagtgat agagatcgtc gacgagct 582207PRTEscherichia coli 2Met Ser
Arg Leu Asp Lys Ser Lys Val Ile Asn Ser Ala Leu Glu Leu1 5 10 15Leu
Asn Glu Val Gly Ile Glu Gly Leu Thr Thr Arg Lys Leu Ala Gln 20 25
30Lys Leu Gly Val Glu Gln Pro Thr Leu Tyr Trp His Val Lys Asn Lys
35 40 45Arg Ala Leu Leu Asp Ala Leu Ala Ile Glu Met Leu Asp Arg His
His 50 55 60Thr His Phe Cys Pro Leu Glu Gly Glu Ser Trp Gln Asp Phe
Leu Arg65 70 75 80Asn Asn Ala Lys Ser Phe Arg Cys Ala Leu Leu Ser
His Arg Asp Gly 85 90 95Ala Lys Val His Leu Gly Thr Arg Pro Thr Glu
Lys Gln Tyr Glu Thr 100 105 110Leu Glu Asn Gln Leu Ala Phe Leu Cys
Gln Gln Gly Phe Ser Leu Glu 115 120 125Asn Ala Leu Tyr Ala Leu Ser
Ala Val Gly His Phe Thr Leu Gly Cys 130 135 140Val Leu Glu Asp Gln
Glu His Gln Val Ala Lys Glu Glu Arg Glu Thr145 150 155 160Pro Thr
Thr Asp Ser Met Pro Pro Leu Leu Arg Gln Ala Ile Glu Leu 165 170
175Phe Asp His Gln Gly Ala Glu Pro Ala Phe Leu Phe Gly Leu Glu Leu
180 185 190Ile Ile Cys Gly Leu Glu Lys Gln Leu Lys Cys Glu Ser Gly
Ser 195 200 205
* * * * *