Note: Descriptions are shown in the official language in which they were submitted.
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ADENOVIRUS E1-COMPLEMENTING CELL LINES
FIELD OF THE INVEN~'ION
This invention relates to novel cells and methods for use in propagating E 1-
deleted adenoviruses.
CROSS-REFERENCE
This application is a continuation-in-part of serial number 08/658,961 filed
May
31, 1996.
BACKGROUND OF THE INVENTION
1 U The majority of adenoviral vectors used in gene therapy applications were
designed to have deletions in the E1 region of the adenovirus 5 (Ad5) genome.
The E1
region, not including region IX, consists of 9% of the left end of Ad5 ( 1.2 -
9.8 map
units), and is subdivided into two regions, E 1 A and E 1 B, each one coding
for several
proteins. Expression of ElA/E1B is required for virus replication and for
expression of
all other Ad5 proteins (E2-E4, Late Proteins; Ginsberg, H.S. The Adenoviruses.
Plenum
Press, New York. p.46-67( 1984). Deletion of E 1, therefore creates a
replication-
incompetent virus that, in theory, is silent for expression of all Ad5
proteins and
expresses only the transgene of interest. Deletion of E 1 A and E 1 B is also
of interest for
safety reasons, since these two proteins, in combmat~on, have been implicated
in
2U oncogenic transformation of mammalian cells (Graham, et al., In Cold Spring
Harbor
Svmp. Quant. Biol. ~ p. 637-650 (1974); Van Der Eb, et al., Gene 2_, p.l 15-
132 (1977);
' McKinnon, et al., Gene 19, p. 33-42( 1982). All of the Class I adenovirus
vectors used to
date in human clinical trials, are deleted for E1.
E1 deficient adenoviral vectors are propagated in an Ad5 helper cell line
called
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293 (Graham, F.L. and Smiley, J, J. Gen. Virol. ~6_, p.54-72 (1977). 293 cells
were
derived by transforming human embryonic kidney cells with sheared fragments of
Ad5
DNA. Genomic analysis revealed that 293 cells contain four to five copies per
cell of the
left 12% of the viral genome (including the entire E1 region) and
approximately one copy
per cell of 9% of the right end, the E4 region (Aiello,L.,et al, Virology 94,
p.460-469
(1979). While 293 cells are very efficient at producing high titers of E1-
deficient
adenovirus, they have the disadvantage that, due to the presence of Ad5
sequences
besides E 1 integrated into the 293 genome, recombination can occur with
sequences in
the El-deficient adenovirus vector causing the production of E1-containing,
replication-
competent adenovirus (RCA). Depending on how early a passage the aberrant
recombination event occurs during the amplification and propagation of the E1-
deficient
adenovirus, and which passage is used for large-scale production of the
adenovirus stock,
production of RCA in 293 cells can present severe ramifications for the safety
of human
gene therapy trials (Lochmuller, H., et al., Numan Gene Therapy 5_, p. 1485-
1491 ( 1994).
In addition to production of RCA, recombination in 293 cells can also cause
deletions and
rearrangements that affect transgene expression, thereby decreasing the titer
of functional
adenovirus particles. Recently, cell lines have been developed using defined
Ad5 DNA
fragments, including the E1 region; however these cell lines contain
significant sequence
overlap with homologous sequences in the E1-deleted adenovirus vectors, thus
allow ng
for undesirable homologous recombination events and the possibility for
generation of
RCA (Fallaux, et al., Human Gene Therapy 7 , p. 215-222 (1996); Imler, et al.,
Gene
Therapy 3_, p. 75-84 ( 1996).
In this invention, a series of cell lines have been generated containing only
the
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minimal E1 gene region for the complementation of E1-deleted adenoviral
vectors, in the
absence of RCA.
' SUMMARY OF THE INVENTION
This invention encompasses a series of helper cell lines for the
complementation,
amplification, and controlled attenuation of E1-deleted adenovirus. These cell
lines are
advantageous because they can complement adenovirus El gene deletions without
production of replication competent adenovirus (RCA). A preferred embodiment
is an
A549E1 cell line that contains only the Ad5 E1 DNA sequences sufficient for
complementation of E1-deleted adenoviral vectors without sequences that
overlap with
the adenovirus vector. In a preferred embodiment, the El DNA sequences
comprise EIA
and E 1 B genes.
In another aspect, the present invention. embodies methods for selectively
propagating mini-adenovirus without generating RCA, by transfecting an A549E I
cell
line with DNA sequences that encode a polypeptide sufficient for packaging
attenuation
of E1-deleted helper virus. In a preferred embodiment, the polypeptide
comprises Cre
recombinase. In another prefer embodiment, the polypeptide comprises TetR-
KR.AB.
BRIEF DESCRIPT10N OF THE DRAWINGS
FiEure 1 is a diagram indicating the structure of adenovirus sequences in a
typical E I -
deleted Ad helper virus (top line), in 293 cells (ref.5), and in other E1-
containing cell
lines, including 911 cells (ref.8) and A549E1-68 cells (this invention); and
how
recombination between homologous adenovirus sequences occurs to generate a
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replication-competent adenovirus (RCA).
Figure 2 is a diagram of the CMV-E1 mammalian expression vector.
Figure 3 is a Southern blot analysis of 6418' A549E1 clones.
FiEure 4A is a Western blot analysis of ElA protein expression in A549 cells,
293 Cells,
S and A549E1-68.
Figure 4B is the metabolic 'SS labeling and immunoprecipitation of EIB
proteins in
A549 Cells, 293 Cells and 4 single-cell clones derived from A549E1-68.
Figure 5 is a representation of the E1-deleted adenovirus vector, Ad5-CA-GFP.
Figure 6 is an agarose gel analysis of 40 PCR reactions using ElA-specific
primers for
detection of RCA.
FiEure 7 is a diagram of a system for the attenuation of helper virus with a
loxP-modified
packaging signal.
Figure 8 is a diagram of the pCMV-Cre-Puro vector.
FiEure 9 is a diagram of the pBS/loxP-stop/MCLpA vector.
i ure 10 is a bar graph depicting luciferase expression in both control cells
(A549E1-
68) and cell lines expressing the TetR-KRAB protein, following transient
transfection
with the pTet07-CM V-L test vector.
DETAILED DESCRIPTION OF THE INYENTIOn
This invention provides cell lines that can complement E1-deleted adenovirus
without the disadvantage of undesirable recombination and RCA. These cell
lines are
obtained by cloning and expressing in the A549 cell line only those sequences
that are
required for E 1 complementation and excluding from the cell line all other
Ad5
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sequences that have homology to the vector and could cause recombination to
produce
RCA.
Currently 293 cells are used to propagate E1-deleted adenovirus. However, 293
cells harbor DNA fragments that make up 21 % of the Ad5 genome. 293 cells
therefore
5 have significant sequence homology (outside of the E1 region) that overlaps
with
sequences in the adenoviral vectors, which can allow for homologous
recombination
events to produce a wild type adenoviral particle. F1G. I shows the structure
of
adenovirus sequences in 293 cells versus other E1-containing cell lines
(including
A549E 1 cells), and how recombination between homologous adenovirus sequences
occurs to generate a replication-competent adenovirus (RCA). Signifecant
homology
(more than 1000 base pairs) exists between Ad sequences 3' to the E1 region in
conventional adenovirus vectors, and sequences integrated into the helper cell
genome of
293 cells and 911 cells. Reciprocal recombination across these homologous
regions can
result in the generation of a wild-type, E1(+), replication-competent
adenovirus (RCA).
This invention embodies the cloning of an ElA and E1B-encoding DNA fragment
from Ad5 into a mammalian expression vector. This E 1 vector was stably
transfected
into human A549 cells to produce an E1 expression cell line. The genome of a
representative cell line. A549E 1-G8, contains no sequence overlap with
sequences present
in the EI-deleted Ad helper virus and thus, recombination to produce RCA is
not possible
(FIG. l ). Characterization of this A549E1 cell line demonstrated the
production of E 1 A
and E 1 B proteins, high infectivity with adenovirus vectors, complementation
of E 1-
deleted adenovirus to produce high-titer virus stocks, as well as, the lack of
production of
replication-competent adenovirus (RCA).
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Further embodiments of the invention are described for the production of 2nd
generation A549-E1 complementing cell lines which, in addition to producing
El, also
produce proteins required for further manipulation of adenoviral vectors, thus
providing a
novel series of RCA-free adenovirus helper cell lines, as tools for novel
virus production.
The first example is an A549E1 cell line which expresses the Cre recombinase.
This
invention provides a novel EI-deleted helper virus whose packaging signal is
flanked by
loxP sites, and when this helper virus is propagated in an A549E1 cell line
expressing
Cre, the packaging signal is deleted by excision, thus attenuating helper
virus packaging
and enriching for packaging of mini-adenovirus (Ad5 virus which is devoid of
all viral
protein-coding sequences). This is advantageous because during the production
of
helper-dependent, mini-adenovirus it becomes necessary to attenuate helper
virus
packaging in order to enrich for the mini-virus. 293-Cre cells have been
generated for
this purpose (Parks, R. , et al., P.N.A.S. 93, p.13565-13570 (1996), however,
A549E1
Cre cells have an advantage in that they would perform this task in an RCA-
free
environment.
A further embodiment of the present invention includes an A549-E 1
complementing cell line which expresses the TetR-KRAB fusion protein, which
would be
used to amplify, and control the packaging efficiency of an E1-deleted helper
virus whose
packaging signal has been modified to contain multiple tetracycline operator
(tet0) sites.
When this helper virus is propagated in an A549E1 cell line expressing TetR-
KRAB, the
repressor binds to the tet0 sequence and specifically attenuates helper virus
packaging,
thus enriching for packaging of the mini-adenovirus which has a normal, wild-
type
packaging signal. Packaging of the helper virus can be restored by growing the
cells and
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virus in the presence of tetracycline, which binds to the tet-KRAB repressor
causing its
dissociation from the tet0/packaging signal and a reversal of packaging
repression.
Detailed examples involving the derivation, characterization, and applications
of these
' El-complementing helper cell lines are described in the following sections.
le 1
Construction of the ElAlEIB vector
To generate an expression vector which harbors only the E 1 A/E 1 B sequences
required for complementation, a 3.1 kb DNA fragment coding for Ad5 E 1 A and E
I B
genes was cloned in two pieces, sequentially, into the superlinker vector,
pSL301
(Invitrogen). First, an 881 by Afl III to XbaI fragment (Ad5 base pairs 462-
1343) was
cloned from pBRXadSKpnICI (a subclone of pJMl7) into pSL301 (Afl III/XbaI).
Second, a contiguous 2194 by Xbal to Afl II (Ad5 base pairs 1343-3537) was
cloned
from pBRXadSXhoIClinto the same vector. The resultant 3075 lip E1 fragment (in
pSL301 ) contains the TATA box and RNA cap site for E 1 A, E1 A coding
sequence,
complete E I B promoter, and E 1 B coding sequence, including the stop codon
for E I B p55
protein, but not including region IX. The 3075 by All Ill - Afl II ElA/EIB
fra~nent
(Ad5 base pairs 462-3537) was isolated, blunt-cndod with Klcnow enzyme, and
blunt-end
ligatcd into the EcoRV site of the mammalian expression vcxlor, pCDNA3
(Im~itrogcn).
under control of the CMV promoter/enhancer. This process generated an Ad5-E1
. expression vector, pCMV-E1 (F1G.2).
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Egam~le 2
Generation and characterization of the El cell line
This CMV-E1 expression plasmid, (FIG. 2), was transfected using Lipofectamine
(GibcoBRL) into A549 human lung carcinoma cells (ATCC CRL 185) and G418R
colonies were isolated. Single-cell clones were screened for functional
ElA/E1B
expression. An E1-deleted adenovirus containing a green florescence protein
(GFP)
expression cassette, Ad5 CA-GFP, was used to infect the A549-E1 clones. Three
days
post-infection, clones were screened for production of El-complemented Ad5 CA-
GFP
adenovirus by visual examination for cytopathic effect (CPE). One clone,
A549E1-68,
displayed 100% CPE in 3 days, similar to that observed for 293 cells. The
clear area in
the center of the plaque is evidence of CPE caused by E1-complemented virus
amplification. This clone also showed high infectivity, in that virtually 100%
of the cells
fluoresced green 24 hours post-infection. The high infection rate and rapid
generation of
CPE induced in this cell line is strong evidence that functional ElA/E1B
proteins are
1 S being produced that are capable of promoting replication and amplification
of the E1-
deleted Ad5-CA-GFP virus. The A459E1 cell was deposited at the American Type
Tissue Culture Collection (ATCC) under 1hc Budapest Treaty on January 15, 1998
as
ATCC Designation CRL-12458 (viability confinmcd January 20. 1998).
FIG. 3 shows a Southern blot using an EI sequence-specific DNA probe. This
assay demonstrated the presence of the CMV-E1 transgene in A549E1-68 (Lane 4),
and a
subclone of A549E1-68 (E1-68.3), but not in the parental A549 cell line (Lane
2).
Sequences hybridizing with the E1-specific probe were also observed in 293
cells as
expected since they complement E1-deleted adenovirus (Lane 3). The morphology
of the
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E1-transfected cells was significantly different from the parental A549 cell
line. A549
cells at sub-confluent density grow as distinct single cells with an
elongated, fibroblast-
like morphology, whereas the E1 cell line A549E1-68 grows as colonies of cells
with a
' more cuboidal morphology. A549E1-68 was also compared with 293 cells for
production
S of E1-deleted adenovirus (Ad5 CA-GFP) by plaque assay and found to produce
an
equivalent titer of complemented virus (7 x l0y pfu for A549E1-68 vs. 9 x
10° pfu for
293).
FIG. 4A shows a Western blot analysis using an ElA specific antibody (M73,
Oncogene Science). This antibody detected two ElA-specific bands with apparent
molecular weights of 46kd and 42kd in the A549E1-68 cell line (lane 3),
corresponding
to products expected from ElA 13S and 12S mRNAs (Ginsberg, 1984), and
identical in
size to those observed in 293 cells (lane 2). These ElA-specific bands were
not detected
in parental A549 cells (lane 1 ). FIG. 4B shows the immunoprecipitation of
metabolically-radiolabeled proteins by a monoclonal antibody specific for E1B
p55.
A549E1-68 produced an immunoreactive band of approximately 55 kd (lane 3) that
was
not detected in parental A549 cells. This 55 kd. E 1 B-specific band, as well
as secondary
background bands, were observed in 293 cells also (lane 2). Extra "background"
bands
found in both experimental and control lanes have been observod by other
authors and arc
attributed to co-immunoprccipitation of a variety of proteins includinb,
cyclins, p53, and
Rb. It is clear that A549E1-68 not only expresses ElA and E1B, but that they
are
functional, since this cell line can complement for production of high titer,
E1-deleted,
recombinant adenovirus.
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Ezam~le 3
EI-deleted adenovirus produced in A549E1 cells is RCA free
To prove that this new Ad5 helper cell line can complement without production
of
RCA, a series of PCR RCA assays were performed following amplification in
A549E1
S cells of the E1-deleted Ad5-CA-GFP adenovirus vector. The Ad5-CA-GFP vector
is
illustrated in FIG. 5. It contains a transcriptional control element
consisting of the CMV
enhancer and the (i-actin promoter and the deletion of El sequences includes a
lack of
Ad5 DNA through base pair 3550. Since the ElA/E1B complementing region
contained
in the A549E1 cells extends only to base pair 3537, there is no overlapping
sequence
10 homology to allow RCA production. EI-deleted vectors with smaller deletions
may still
contain some E1 DNA and should be avoided, as they could still allow RCA to
occur at a
low frequency.
For the PCR RCA assay, Ad5-CA-GFP virus was serially propagated through 20
passages on A549E1-68 cells. Following serial propagation and virus
amplification,
Ad5-CA-GFP virus DNA was isolated by freeze-thaw lysis, and PCR was performed
usinb primers specific for either the ElA region or the E2B region.
Amplification of an
880 by E2B product serves as a PCR positive control, while the presence of a
1086 by
E 1 A-specific product is evidence that an E 1 (+) replication-competent
adenovirus (RCA)
has been produced during amplification of the E1 (-) Ad5-CA-GFP. 20 ug of Ad5-
CA-
GFP virus DNA (equivalent to 1 x 10'° virus particles), obtained from
amplification in
A549E1 cells, was divided into 40 PCR reactions and tested for RCA using the
ElA
primers (FIG. 6). For both top and bottom panels of FIG. 6, lane 1 contains 1
kb DNA
markers, lane 2 contains wild type Ad5 virus DNA, lane 3 consists of PCR of
Ad5-CA-
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11
GFP virus DNA (E1-) isolated from 20+ passages on A549E1-68 cells using ElA
and
E2B specific primers (positive control), and lanes 4-20 consist of PCR of Ad5-
CA-GFP
virus DNA (E1-) isolated from 20+ passages on A549E1-68 cells, using ElA-
specific
primers only. No 1086 by EI region specific PCR fragments were detected in any
of the
reactions indicating that no RCA was present in the virus prep.
A second, CPE-based RCA assay was performed by amplifying EI-deleted
adenovirus (Ad5-CA-GFP) on A549E1-68 cells and testing the amplified virus by
passaging, on normal A549 cells (don't make E1) for production of EI-
containing RCA.
Plaque formation (CPE) on a monolayer of normal A549 cells would provide
evidence
for the production of wild-type (EI +) virus during amplification on the El
helper cell
line, A549E1-68. 2 x 10'° EI (-) virus particles (amplified using
A549E1-68) were used
to infect each of five 150mm plates of normal A549 cells (I x 10" panicles
total). No
CPE or single plaques were detected after 8 days on any of the A549 plates,
indicating
the absence of any E1 (+) RCA virus in the A549E1-68-amplified virus prep.
Therefore,
using two sensitive assays for detection of RCA, no wild-type recombinant EI
(+) virus
was detected, supporting the utility of this cell line for the amplification
and large-scale
preparation of El-deleted adenoviral vectors in the absence of RCA.
Exam
Generation oJan A549E1 Cre Cell Line
- A 2nd generation E I -complementing cell line was generated using the A549E
1-
68.3 clonal line for transfection with Cre recombinase. This cell line will
both
complement E1-deleted adenovirus vectors and mediate the excision of sequences
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surrounded by loxP sites. Our primary use for this cell line is to further
attenuate
packaging of an Ad5 helper virus, whose packaging signal is flanked by two
IoxP sites
(FIG. 7), in order to enrich for packaging of the desired E1-deficient, mini-
adenovirus
vector. 293 cells expressing the Cre recombinase were generated for a similar
purpose by
S Parks et al. (P.N.A.S. 93:13565-13570), and were shown to increase the titer
of E1-
deleted vector virus 10 fold per passage, demonstrating the overall utility of
this system
for removal of helper virus. The A549E1-Cre cell line described in this
invention will
not only attenuate helper virus packaging in a similar fashion, it also has
the advantage
that any adenovirus produced will be free of deleterious RCA.
As a first step towards the production of the A549E1-Cre cell line, a Cre
expression vector was constructed. A 1440 by SV40 promoter-puromycin cassette
(for
selection in Neon A549E1 cells) was cloned into a unique EcoRl site of the CMV-
Cre
vector (pBS185, GibcoBRL) to generate pCMV-Cre-Puro (FIG. 8). The pCMV-Cre-
Puro vector was transfected by electroporation into A549E 1-68 cells, and
puromycinR
("puroR") clones were isolated. These puroR clones were then screened for
expression of
functional Cre recombinase. The plasmid pBS/loxP-stop/MCLpA contains a lacZ
cassette that is non-functional due to the presence of a stop colon (FIG. 9).
This stop
colon is surrounded by loxP sites, such that the propagation of this vector in
a cell line
producing Cre would excise the stop signal and activate the IacZ gene. The
pBS/loxP-
stop/MCLpA vector was transiently transfected into each of the A549E1-Cre
clones, and
after 24 hours. the transfected cells were fixed and stained with X-Gal. LacZ
expression
of parental A549E1-68 cells (no Cre) was compared to lacZ expression in seven
different
puro'A549E1-Cre clones. Expression of lacZ (due to expression of Cre) was
observed as
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blue cells, at a frequency ranging from 1 % to 50% in 20/26 puroR clones. This
range of
LacZ-expressing cells is most likely a reflection of the transient
transfection efficiency of
the different puroR clones with the pBS/loxPstop-MCLpA vector, although it
could also
reflect variations in Cre recombinase expression in different cell lines.
Western blot
analysis using an anti-Cre antibody (Pharmingen), confirmed the presence of
the 35 kd
Cre protein in these cell lines. Experiments to assess the attenuation of Ad
helper virus
containing a packaging signal flanked by two loxP sites are in progress.
Example 5
Generation oJan A549E1 cell line expressing TetR-KRAB
A highly conserved, 75 amino acid protein called the Kruppel-associated box
(KR.AB) was recently isolated by Margolin, et al. (P.N.A.S. ~, p.4509-4513
(1994)).
The KRAB box is a member of the Kox-1 family of human zinc finger proteins and
was
subsequently shown to be a strong transcriptional repressor. By fusing the
KRAB
domain from Kox-1 to the Tet repressor derived from TnlO of Escherichia coli,
a hybrid
protein was generated, TetR-KRAB, which allows tetracycline-controlled
silencing of
eukaryotic promoters (Deuschle, et al., Mol. and Cell. Biol. ]s,5, p.1907-1914
(1995). The
TctR-KRAB repressor binds to tet0 soquenccs present in a transcriptional
control region
and represses transcription of genes placed as far as 3 kb downstream.
The present invention describes a system for tetracycline-controlled
inhibition of
helper virus packaging, comprising multiple tet0 sequence in the helper virus
packaging
signal sequence, and an E1 helper cell line that constitutively expresses the
TetR-KRAB
protein. The helper virus is still capable of replicating and providing all
the necessary
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proteins, in traps, required for replication of the miniAd vector, however,
its packaging is
attenuated due to binding of the TetR-KRAB protein to the tet0 sites in the
packaging
signal. The overall goal is to hinder or repress helper virus packaging, thus
enriching for
vector virus packaging. This packaging repression is reversible, since in the
presence of
S tetracycline, the TetR-KRAB repressor dissociates from the tet0 sequences,
and
packaging is restored. Details of this tet0-controlled helper virus were
presented in an
earlier patent application (Serial No. 08/658,961, filed May 31, 1996).
The TetR-KR.AB expressing cell line was derived using the A549E1-68 helper
cell line described in Example 2. A549E1-68 cells were transfected with a TetR-
KR.A.B
gene under control of the CMV promoter (see Deuschle et al., Mol. Cell. Biol.
15 p.
1907-1914 (1995). The TetR-KRAB vector also contains a hygromycin resistance
gene
for selection in mammalian cells. A test vector (see Deuschle, et al., Mol.
and Cell. Biol.
_1~,, p.1907-1914 (1995)) that has a luciferase reporter gene under control of
the CMV
promoter fused to a TetO sequence was used for transient transfection into
cells lacking a
TetR-KRAB repressor. This transfection results in high level expression of
luciferase,
whereas transfection into A549E1 cells expressing the TetR-KRAB protein will
result in
the repression of luciferase due to binding of the repressor to tet0 sites in
the test vector.
Hygromycin-resistant A549E1-TetR-KRAB clones were transfectcd with pTetO-CMV-L
by electroporation and each clone was split into two wells of a 6-well plate.
24 hours
post-transfection, cells from one duplicate well were refs with medium
containing
tetracycline, and the other duplicate well in medium without tetracycline.
After another
24 hours, cells were lysed and assayed for luciferase expression using a
Promega
Luciferase Assay Kit. Two hygroR A549E1 clones {TKE-9 and TKE-12) demonstrated
a
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4 to 6 fold repression of luciferase reporter activity when grown in the
absence of
tetracycline versus cells grown in media containing Tet, indicating expression
of the
TetR-KR.AB repressor protein in the cells (FIG. 10}. These A549E1-TetR-KRAB
cell
lines will be used to test attenuation of the TetO-controlled Ad helper virus.
5 These examples are intended to illustrate the present invention and are not
intended to limit it in spirit or scope.
