Note: Descriptions are shown in the official language in which they were submitted.
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Title: MODIFIED ADENOVIRAL VECTORS FOR USE IN GENE THERAPY
The present invention relates to the field of human gene
therapy, in particular to gene therapy vehicles with reduced
expression of viral genes, more specifically with a reduced
immunity. The invention provides novel expression vectors and
complementing cell lines, including means and methods to
produce such vectors and cell lines, and applications of such
vectors, cell lines and methods in human gene therapy
protocols.
The development of human gene therapy for the-treatment of
to inherited and acquired disorders requires gene transfer
vectors capable of safe and effective delivery and expression
of therapeutic genes into target cells. The actual method to
introduce and express the genetic material into the target
cells of a patient is a key component in every gene therapy
protocol. Several different gene transfer systems are
currently being employed to introduce therapeutic genes into
somatic cells.
Non-viral gene systems for in vivo delivery into target
cells include a variety of DNA-mediated methods, like direct
2o injection of naked DNA and particle bombardment. To overcome
the limitations of these DNA-based delivery methods (low
transfer efficiency, and cell toxicity), alternative systems
have been developed utilizing the entrapment of the DNA in
vesicles (like liposomes), or binding of the DNA to synthetic
2s conjugates (like e.g. transferrin-polylysine). Such
conjugate-DNA complexes can be delivered via receptor-
mediated endocytosis.
Viral gene-transfer systems are based on the natural
capability of viruses to deliver their genes in mammalian
3o cells. The high gene-transfer efficiency of viruses has led
to the development of viral vectors in which part of the
viral genome has been replaced by a transgene to be
introduced into eukaryotic cells. The most commonly used
viral systems are based on retroviruses, adenoviruses (Ad)
35 and adeno-associated viruses. Each of these viral delivery
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systems has its own characteristics in terms of efficiency of
gene delivery, integration capability, maximum insert size of
the recombinant (recombinant) gene, vector yields, stability
of expression, etc. Such characteristics may determine the
suitability of a certain delivery system for a specific gene
therapy protocol (Bout, 1997). Retroviruses and adeno-
associated viruses have been a focus for development because
of their capacity to stably integrate DNA sequences into
chromosomes of the target cells. The advantage of
io adenoviruses is their ability to mediate efficient expression
of the transgene in a variety of cells, including post-
mitotic and/or non-dividing cells, as well as the ease with
which these viruses can be propagated and purified to very
high titers.
i5 Inherent to all gene transfer methods is the
presentation of foreign antigenic material on the target
cell, derived from the vehicle and/or from the transgene
encoded product. The presentation of foreign antigens usually
elicits a response of the immune system; immune responses
2o directed against the gene therapy vehicle and/or the
transgene product may lead to inflammation, elimination of
the transduced cells, and difficulties with re-administration
of the vector due to neutralizing activity against the
vehicle. As a consequence, the clinical application of gene
z5 therapy vectors can be impeded by the potent host immune
response to the vector, which limits the duration of its
effects. Immune effectors have been identified as cytotoxic T
lymphocytes (CTLs), which destroy vector-transduced cells, as
well as B cells; which secrete neutralizing antibodies that
3o can block repeated gene transfer.
CTLs continuously monitor the cells and tissues of the body
in search of cells synthesizing foreign or abnormal proteins.
The recognition of virus-infected cells by CTLs requires
35 fragments (i.e. peptides) of foreign antigens that are
presented at the ce111surface in association with class I
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molecules of the major histocompatibility complex (MHC; for a
recent review see Ploegh et al., 1998). The majority of these
peptides are generated in the cytosol of virus-infected cells
by degradation of poly-ubiquitinated viral proteins. The
s resulting viral peptides are transported from the cytosol to
the endoplasmic reticulum (ER), through the action of the ATP
dependent transporter for antigen presentation (TAP) complex.
In the ER, MHC class I heavy chains assemble with (32
microglobulin and peptide into stable hetero-trimeric MHC
io class I complexes that are transported via the secretory
route to the plasma membrane. Their expression at the cell
surface enables CTLs to play their decisive role in the anti-
viral defense.
In the first generation of adenoviral vectors for gene
i5 therapy, the E1 region was replaced by foreign genetic
information; e.g., the therapeutic gene. The absence of E1
renders the recombinant virus replication defective. Because
E1 has been reported to trigger the transcription of other Ad
genes, it was previously thought that E1-deleted vectors
2o would not express any Ad genes. However, it has been shown by
others and us that early (e. g. E2A, E2B and E4) and late
(e. g. fiber, hexon and penton-base) genes are still
residually expressed from such vectors. This residual Ad gene
expression is due to background replication of the
2s recombinant viral genome and the background activity of the
promoters driving the transcription of the respective Ad
genes (Yang et al., 1994; Lusky et al., 1998). This means
that the delivery of a therapeutic gene into target cells
using El-deleted Ad vectors results in expression of the
3o therapeutic gene as well as of the viral genes. Eventually,
this will lead to the presentation of viral peptides by MHC
class I complexes followed by a cytotoxic immune response
against the transduced cells. It has been shown that CTLs
directed against the transgene product as well as against the
3s Ad gene products are activated following administration of
the vector into immune-competent animals (Song et al., 1997;
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Yang et al., 1996). The activated CTLs subsequently eradicate
the transduced cells from the recipient.
In order to reduce the residual expression of viral
genes from recombinant Ad vectors, we have generated Ad
vectors that are deleted for the E1 and E2A region. To
complement the deletions of E1 and E2A, we have generated an
E1 + E2A complementing cell line for the manufacturing of
high titer E1/E2A deleted recombinant Ad vector batches, as
described herein. Due to the toxicity of the E2A encoded DNA
1o binding protein (DBP), it is difficult to generate a cell
line that constitutively expresses E2A. Therefore, we have
made use of a mutant E2A gene, derived from H5ts125, that
encodes a temperature sensitive (ts) DBP (van der Vliet,
1975). The tsDBP is functionally active at the permissive
temperature (32°C), whereas it is nonactive at the
nonpermissive temperature (39°).~ In addition, tsDBP is not
toxic at the nonpermissive temperature. Thus, we established
a new cell line, designated PER/E2A, that constitutively
expresses high levels of the tsE2A gene. This cell line can
2o easily be cultured at the nonpermissive temperature. V~Ihen
functionally active DBP is needed, e.9., for replication of
E2A-deleted Ad vectors, the cells can be incubated at the
permissive temperature. The PER/E2A cell line and the E1/E2A
deleted vectors were designed such that overlap between the
2s Ad sequences in the cell line, i.e. E1 and E2A coding
adenoviral sequences, and sequences in the recombinant Ad
vector was excluded, at least to the extent that may lead to
homologous recombination between vector DNA and adenoviral
sequences present in the complementing cell line, which could
30 lead to the formation of reverted viruses that have
recaptured the E1 and/or E2A genes.
The deletion of the E2A gene eliminated the residual
expression of E2A and the expression of Ad late genes, e.g.,
penton-base and fiber (Figure 1A). However, these vectors
3s still expressed significant amounts of the E4, e.g. E4-orf6,
and E2B genes, e.g., pTP (Figure 1B). Transcription of E4 may
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even be up regulated in the absence of DBP since the E4
promoter is a natural target for repression by DBP (Blanton
and Carter, 1979; Nevins and Winkler, 1980; Chang and Shenk,
1990). Hence, cells infected by E1/E2A deleted vectors still
5 produce and present non-self antigens, when delivered to
humans and may be eradicated from the recipient by the immune
system. In order to solve such problems the present invention
provides modifications to E2B and/or E4 regions or regions
controlling the E2B and/or E4 regions in adenoviral vectors
to and/or in the complementing cell lines therefor.;
E4 constitutes approximately 10°s of the total length of
the Ad genome. Several differentially spliced mRNAs are
synthesized from the E4 region during infection and are
predicted to code for seven different polypeptides, six of
i5 which have been identified in infected cells.
Genetic studies have shown that E4 encoded proteins have
an important function in virus growth in cultured cells since
mutant viruses that lack the entire E4 region have a severe
defect in replication (Weinberg and Ketner, 1986; Huang and
2o Hearing, 1989). Such E4 lacking viruses display defects in
viral DNA replication, viral late mRNA accumulation, viral
late protein synthesis, and the shut-off of host cell protein
synthesis. Although the exact function of all individual E4-
encoded polypeptides has not been defined to date,
25 mutagenesis of individual open reading frames (orfs) has
shown that multiple products encoded by E4 are functionally
compensatory. Thus, it was found that either the E4-orf3 or
the E4-orf6 product is prerequisite for virus replication in
cultured cells whereas the other E4 products are dispensable
30 (Huang and Hearing, 1989).
E4-orf3 as well as E4-orf6 encoded proteins are
(independently) involved in post-transcriptional processes
that increase viral late protein synthesis. They do so by
facilitating the cytoplasmic accumulation of the mRNAs
35 encoding these proteins (Sandler and Ketner, 1991). Moreover,
they maintain the nuclear stability of unprocessed pre-mRNAs
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transcribed from the major late promoter, presumably by
affecting the splicing of late RNAs. This leads to an
expansion of the pool of late RNAs available for maturation
and transport to the cytoplasm. In addition, the E4-orf6
s encoded 34 kDa protein forms a complex with the E1B 55 kDa
protein that selectively increases the rate of export of
viral late mRNAs from the nucleus. The E4orf6-34 kDa/E1B-55
kDa complex is located in so called viral inclusion bodies,
the region where viral DNA replication, viral late gene
io transcription and RNA processing occur (Pombo et al., 1994).
Finally, both the E4-orf6 and E4-orf3 encoded proteins are
required for Ad DNA synthesis.
Proteins encoded by E4 also interact with cellular
proteins and antagonize cellular processes. For example, E4
15 protein products are involved in controlling the cellular
transcription factor E2F as well as the phosphorylation of
cellular (and viral) proteins. Moreover, both E4-orfl and E4-
orf6 products have oncogenic potential. The E4-orf6 protein,
either alone or in a complex with the E1B-55-kDa protein,
2o binds the cellular protein p53 thereby blocking its potential
to activate the transcription of tumor-suppressing genes
(Dobner et al., 1996; Moore et al., 1996). As a result, the
E4-orf6 protein may prevent the induction of apoptosis by
p53.
2s Taken together, disrupting or deleting the E4 function
from recombinant Ad vectors may further reduce viral genome
replication and expression of early and late viral genes.
This may, in turn, diminish the antigenicity of the vectors.
In addition, vectors from which the E4 function is impaired
3o can be considered safer than E4-containing vectors because
they do not express E4-encoded proteins that can induce
oncogenesis or which are toxic to the host cell.
It is important to note, however, that the impairment of
the E4 function within the Ad vector backbone can influence
3s the activity of the promoter that drives the expression of
the transgene. For example, it has been reported that the
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activity of either the cytomegalovirus (CMV) promoter or the
Rous sarcoma virus (RSV) promoter is down-regulated when the
E4 region is largely or completely deleted from the Ad vector
(Armentano et al., 1997; Brough et al., 1997; Dedieu et al.,
s 1997). Thus, although the transduced cells and the vector DNA
may persist, the expression of the transgene from the CMV or
the RSV promoter is only transient when an E4-specific
factors) is absent. The underlying mechanism causing the
silencing of these promoters is unclear, but a recent study
io point to the requirement of E4-orf3 for long-term expression
from the CMV promoter (Armentano, 1999). The same study also
showed that a truncated form of the CMV promoter could
persistently drive expression in the absence of E4. It is yet
unknown whether promoters from genes of mammals (mammalian
is promoters) are influenced by the E4 region when present in
the context of a recombinant Ad vector. It is therefore
highly valuable to determine whether mammalian promoters,
such as the elongation factor-1 alpha (EF-la) or the
ubiquitin-C (UbC) promoter, can mediate persistent expression
2o of the transgene from an Ad vector that does not produce E4.
The early region 2 (E2) of Ad encodes three gene products
that are required for Ad DNA replication. E2 can be divided
into two transcription units, E2A and E2B. Both E2A and E2B
2s are transcribed from the same promoter region, designated the
E2 promoter. At early time points after infection, E2 is
transcribed from the E2-early promoter located at 76 map
units. At intermediate times after infection, the
transcription switches to another promoter, the E2-late
3o promoter located at 72 map units (reviewed by Swaminathan and
Thimmapaya, 1995). Transcription initiation from the E2-early
promoter is strongly induced by the polypeptides encoded by
E1, which is mediated via E2F and jun/ATF transcription
factors. Transcription initiation from the E2-late promoter
35 1S less well understood. This promoter consists of a TATA-
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like sequence, SP-1 binding sites and a CAAT box. The E2-late
promoter is not regulated by proteins of the ElA region.
The E2A region encodes the 72kDa single stranded DNA
binding protein (DBP). It plays a pivotal role in both the
s initiation and elongation of Ad DNA replication (reviewed by
van der Vliet, 1995 and references therein). Briefly, DBP is
thought to increase the affinity of the host cell nuclear
factor 1 (NF1) to the auxiliary region of the inverted
terminal repeat (ITR) of the Ad genome. This, in turn,
to facilitates binding of the pTP/pol complex (see below) to the
core region of the ITR. Secondly, DBP stimulates the NF1
dependent formation of pTP-dCMP, which forms the DNA-
replication initiation complex. Thirdly, DBP facilitates Ad
DNA template unwinding by destabilization of the DNA helix in
is the replication fork. Following unwinding, DBP binds
cooperatively to the single stranded Ad DNA in a non-sequence
specific manner, thereby forming a protein chain at the
displaced strand. This may be the mechanism by which DBP
destabilizes the duplex DNA ahead of the replication fork.
2o Fourthly, DBP prevents intramolecular renaturation of the
ITRs of the displaced DNA strand, whereas it facilitates
intermolecular renaturation of two displaced strands of
opposite polarity, originating from initiation of DNA
replication at different molecular ends. Finally, DBP has a
25 positive regulatory effect on the activity of the major late
promoter, the promoter that drives expression of the Ad late
genes (Chang and Shenk, 1990).
Transcription of E2 gives rise to two E2B-specific mRNAs
that are derived from differential splicing. They encode two
3o polypeptides, the 80 kDa pTP and the 140 kDa DNA polymerase
(pol) that form a stable heterodimer. The pTP/pol complex is
recruited to the ITR by NFl, where it binds to the core
region and forms the pre-initiation complex. Next, the ITR
partially unwinds and the replication-initiation reaction is
3s primed, i.e. a pTP-dCMP coupling takes place that is
catalyzed by pol. This is followed by synthesis of a pTP-CAT
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intermediate at G4-TS-A6 of the ITR. The pTP-trinucleotide
intermediate jumps back to the G1-T2-A3 position of the ITR.
Elongation of the Ad DNA synthesis starts after dissociation
of DNA-bound pTP from pol. The elongation reaction is
s enhanced by DBP, as discussed above, and progeny DNA
accumulates. Finally, the DNA-bound pTP as well as free pTP
are proteolytically cleaved into TP by the Ad protease late
in the infection cycle. The proteolytic maturation of free
pTP destroys its capacity to function as a primer for DNA
1o replication and thus the initiation of new DNA replication
cycles is stopped. Mature TP that is covalently bound to
newly synthesized Ad DNA protects the DNA from exonuclease
activity and is involved in the attachment of the viral DNA
to the nuclear matrix. Finally, DNA-bound TP stabilizes the
i5 incoming pTP/pol heterodimer at the core region of the ITR
during the initiation of DNA replication in the next lytic
infection cycle.
The deletion approach to knock out the viral gene
2o functions from the recombinant Ad vector is difficult to
apply to E2B and E4 for the following reasons. First, since
both E4 and E2B encoded proteins are pivotal for the lytic
infection cycle of Ad, the production of recombinant Ad
vectors that lack these regions would require a complementing
2s cell line that is equipped with expression vectors that
ectopically express, in addition to E1 and/or E2A, at least
E4-orf6 and/or E4-orf3 as well as pTP and pol. Although the
generation of such cell lines may be possible, these cell
lines will normally be unstable. In addition, some of these
3o viral gene products, e.g., E4orf6 34 kDa and E4orf3 11 kDa,
are considered to be highly toxic when constitutively
synthesized in a complementing cell, meaning that the
expression of the corresponding genes needs to be regulated
tightly. This generally leads to complementing cells that do
35 not support manufacturing of high titer batches of the
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respective recombinant Ad vectors (Lusky et al., 1998 and
references therein).
Secondly, the approach to delete viral gene functions
from the recombinant Ad vectors is difficult to apply to E2B.
s Although it is possible to generate cell lines that
constitutively express pTP and pol (Amalfitano et al., 1997),
it is not possible to entirely delete the pTP and pol coding
sequences from the vector genome. This is due to the fact
that other viral regulatory elements and genes are present in
io this area of the viral genome. This area includes the second
and third tripartite leader sequences, the i-leader, portions
of the major-late promoter intronic sequences and the IVa2
gene (Amalfitano et al., 1997). As a consequence, only
partial deletions in the pTP and pol coding sequences can be
i5 made. This yields a substantial sequence overlap between the
remaining sequences in the recombinant adenoviral vector and
the pTP and pol sequences in the complementing cell line.
This, in turn, can lead to homologous recombination and
reversion of the pTP and pol deleted phenotype during virus
2o replication. Therefore, the deletion approach is not suitable
for the production of a homogeneous population of E2B-
crippled Ad vectors.
Thirdly, deletion of the entire E4 transcription
unit is feasible but it seems to impair the expression of the
z5 L5 region encoding the fiber protein (Brough et al., 1996).
This causes a severe reduction in virus yields when an E4
deleted vector is grown in E4 complementing cells. This
defect can be partially solved by introducing a spacer
sequence, e.g., an expression cassette, in place of the
3o deleted E4 sequences (GenVec, US patent 5,851,806).
Fourthly, it should be noted that recombinant Ad
vectors that are deleted of some or even all viral coding
sequences (so-called gutless or minimal vectors) can only be
propagated using a helper Ad that supports replication and
35 packaging of such vectors by providing all the necessary
proteins in trans (Fisher et al., 1996; Hardy et al., 1997;
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Kochanek et al., 1996; Kumar-Singh and Chamberlain, 1996).
Generally, the packaging sequence of such a helper virus is
flanked by lox sites which are targets for the CRE-
recombinase. Hence, the packaging signal is excised from the
s helper vector when the packaging cells (used for production
and packaging of the gutted vectors) express the CRE-
recombinase, thereby preventing the packaging of the helper
vector. This system, however, has serious limitations with
respect to the efficiency of excision of the packaging signal
io and to the low yields of the gutted vector. Therefore, the
crude vector batches produced in this way are contaminated
with helper virus that is formed due to inefficient excision
of the packaging signal. Moreover, the removal of this
contaminating helper virus is laborious and incomplete, which
15 means that it is practically impossible to obtain a helper
virus free vector batch.
A final problem is that extensive deletion of the coding
sequences from the Ad genome renders the virus unstable and
leads to rearrangement of the viral DNA during replication
20 (Parks and Graham, 1997). This is presumably due to fact that
genomes smaller than 75% of the wild-type genome are
inefficiently packaged into virus particles. To circumvent
this problem, the deletion of large parts of the viral genome
has to be compensated by addition of heterologous sequences
2s to increase the net size of the vector genome. Such an
approach has the intrinsic risk of introduction of
unintentional, and perhaps yet unknown cryptic
transcriptional signals and open reading frames within the
vector backbone and increases the risk of (homologous)
3o recombination with the cellular DNA during replication.
The present invention now provides a method for
producing a recombinant adenovirus-like gene delivery vehicle
having reduced expression of adenoviral E2B and/or E4 gene
products in a target cell for gene therapy, comprising
35 generating a recombinant adenoviral vector lacking ElA and
preferably E1B sequences, but having at least the E2B and/or
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E4 sequences encoding products essential for adenoviral
replication, wherein said E2B and/or E4 sequences have been
modified to lead to a reduced expression and/or induced
expression of at least one of said essential products.
Modification in this respect means any change at the nucleic
acid level that diminishes the expression and/or the function
of any of the gene products of the relevant genes, be it by
mutation in a coding sequence or mutation in a regulatory
sequence, whereby the expression is not completely or
io permanently deleted. It also means replacing a regulatory
sequence of any of these genes by one or more inducible
regulatory sequences, be it repressors, transactivation
sites, inducible promoters or any other inducible sequence,
whereby non-leaky ones are preferred. Combinations may be
i5 made to avoid leakage in the non-activated or repressed
state. In a preferred embodiment at least
open reading frame 1, 3 or 6 of E4 is so modified. An
additional advantage of at least some E4 modifications
according to the invention, is that concurrently E2B
2o expression is at least in part reduced in said gene delivery
vehicle. Thus expression of E2B can be reduced directly by
modifying E2B sequences or indirect by modifying E4 sequences
or by both type of modifications. Similarly, expression of E4
gene products can be reduced by modification of E4 sequences
z5 or by modification of E2B sequences or both. As disclosed
herein, open reading frame 1, 3 or 6 of E4 are sequences
that are essential for replication and/or sequences that lead
to toxicity for target cells and/or complementing cells.
Therefor down regulation or induction of such sequences is
3o highly desired. Attenuation is preferably achieved through at
least one mutation in at least an E2B and/or E4 promoter.
Thus the invention also provides a method as disclosed herein
before, wherein said vector further comprises an E2B and/or
an E4 promoter, wherein said E2B and/or E4 promoter are
35 attenuated through a mutation therein. In the alternative or
in combination with the above the invention provides in one
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embodiment a method according as disclosed herein before,
wherein E2B and/or E4 is placed under control of at least
one, preferably synthetic inducible promotor and/or
repressor. Suitable inducible promoters are well known in the
s art. A couple of suitable and preferred ones are disclosed
herein in the detailed description. Highly preferred
inducible promoters are the ones that are described as
synthetic, comprising e.g. an artificial TATA-box and a
sequence capable of being recognized by a prokaryotic or
1o similar transactivation signal. Typically a complementation
cell would then be able to provide said signal through e.g.
an expression cassette introduced therein.
As described below it is preferred that the vector also lacks
a functional E2A region, which can be elegantly provided by a
i5 complementing cell, especially in the form of a temperature
sensitive variant of E2A.
A function of the E4 34 kDa protein can also be attenuated by
inhibiting the binding to E1B 55kD. Thus the invention also
provides a method according wherein said vector lacks a
2o sequence encoding E1B 55kD protein capable of binding an E4
34 kDa gene product.
In order to produce a gene delivery vehicle according to the
invention the vectors according to the invention are
propagated in complemanting cells. Thus the invention also
z5 provides a method as disclosed above, further comprising
transducing a complementing cell with said recombinant
adenoviral vector wherein said complementing cell provides
all functions and/or elements essential for replication of
said recombinant adenoviral vector, which are lacking in the
3o genome of said vector. This is the normal concept for making
gene delivery vehicles, except that it now has the advantages
as disclosed herein by modification of E2B and/or E4
expression. A gene delivery vehicle according to the
invention is defined as any viral particle derived from an
35 adenovirus, a chimaeric adenovirus or comprising adenoviral
elements, capable of infecting cells and delivering a gene
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there to. A chimaeric adenovirus may be a chimaera of two or
more different adenoviruses, manipulated to give good
infection and yet low antigenicity, etc. It may also be a
chimaera of adenovirus with another virus such as AAV or a
s retrovirus, in order to be able to integrate a nucleic acid
of interest into a host cell genome. It may even be only a
fiber or an adenoviral receptor recognising part of an
adenovirus coupled to another virus or non viral vehicle
comprising the vector.
to The invention also provides a method wherein said
complementing cell further comprises all necessary functions
and/or elements essential for producing a recombinant
adenovirus-like gene delivery vehicle comprising said
recombinant adenoviral vector. Here the function of the
15 complementing cell goes beyond replication of the vector and
typically includes packaging of the vectors according to the
invention, which should then typically possess a functional
packaging signal.
As stated herein before, the complementing cell according to
2o the invention preferably also comprises the capability to
provide the induction of the E4 and/or E2B products. Thus the
invention provides a method wherein said cell further
comprises an expression cassette encoding a proteinaceous
substance capable of transactivating the inducible
2s (synthetic) promoter on the vector, preferably a method
wherein said proteinaceous substance comprises a DNA binding
domain from a prokaryote or a lower eukaryote and/or a
transactivator domain. More details are given in the detailed
description. To avoid problems associated with the production
30 of replication competent adenoviruses and/or the production
of transforming activity in the preparation of gene delivery
vehicles it is preferred to apply a method wherein said
recombinant vector and said complementing cell have no
sequence-overlap that leads to homologous recombination
3s resulting in replication competent adenovirus and/or
recombinant adenovirus comprising E1 sequences.
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The invention also provides the vectors obtainable by
methods as disclosed herein. In one embodiment the invention
thus provides a recombinant adenoviral vector lacking ElA and
preferably E1B sequences, but having at least the E2B and/or
5 E4 sequences encoding products essential for adenoviral
replication, wherein said E2B and/or E4 sequences have been
modified to lead to a reduced expression and/or induced
expression of at least one of said essential products, said
vector being obtainable as an intermediate in a method as
Zo disclosed above. Also provided are the adenovirus-like gene
delivery vehicles having reduced expression of adenoviral E2B
and/or E4 gene products in a target cell for gene therapy,
obtainable by a method as described herein. Of course such a
vector and/or vehicle preferably comprises a therapeutic
15 sequence such as a gene encoding a therapeutic protein, an
anti-sense sequence, etc. Such a vector can subsequently be
used to introduce the therapeutic protein, anti-sense
sequence etc. in cells of a patient, for example (but not
limited to) to correct a certain inherited or acquired
2o disorder or for vaccination purposes.
In gene therapy settings one advantage of a recombinant
adenoviral vector of the invention is that expression of a
nucleic acid interest delivered to a host through said vector
is prolonged compared to a adenoviral vectors of the art.
2s This is not only due to improved capabilities to avoid the
host immune system (in or outside a cell) but also to other
factors. Another reason for the observed prolonged expression
is that promoters that are commonly used for the expression
of gene of interest in a gene therapy setting are at least in
3o part protected from shut down of expression in the host. A
non-limiting example of such a commonly used promoter is the
CMV promoter.
35 Detailed description
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The present invention provides means and methods for the
generation and manufacturing of recombinant Ad vectors that
are modified in E2B and/or E4 functions, preferably, said
vectors comprise E1 and/or E2A deletions. For this purpose,
the vector genome is modified in the respective promoter
regions, such that the promoter is only active in a suitable
complementing cell line or only active following a certain
signal in the case of an inducible promoter. The modified
promoter is, on the other hand, inactive under normal
to conditions, and in normal mammalian and/or human cells.
Hence, vectors that possess said modified promoter in the E2B
and/or E4 region do not express the respective transcription
regions in mammalians and/or humans. The promoter
modifications can be made in recombinant Ad vector genomes
that lack E1, or E1 and E2A, or E1 and E3, or E1 and E2A and
E3, or E1 and E4, or E1 and E2A and E4, or E1 and E3 and E4,
or E1 and E2A and E3 and E4. In one aspect the invention
provides the latter four variants in the case of E2B promoter
modification only. Since there is no overlap between vector
2o sequences and adenoviral sequences in the complementing cell
line, reversion of the modified phenotype due to homologous
recombination cannot occur.
Successful replacement of the E4 promoter by a synthetic
promoter has recently been reported (Fang et al., 1997). In
this study, the E4 promoter was replaced by synthetic Gal4
binding sites and the vectors modified in the E4 region could
be efficiently propagated in 293 cells expressing the
appropriate Gal4/VP16 fusion protein. However, replication of
the vectors modified in the E4 region was dramatically
3o impaired in cells that did not express the Gal4/VP16 fusion
protein.
It was assumed impossible by persons skilled in the art
to manipulate the E2 promoters 'because of overlap with the
major late open reading frame L4' (quote-unquote by Rittner
et al., 1997). However, in the present invention, we provide
means and methods to produce vectors that are attenuated in
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17
botheE2 early and late promoter regions, in particular in
vectors that are also deleted for the E2A gene.
For use in gene therapy, it would clearly be
s advantageous to have recombinant Ad vectors available that do
not produce or produce reduced amounts of viral polypeptides
and hence are less immunogenic, but yet retain all
capabilities of the current vectors. The present invention
provides novel recombinant Ad vectors that improve
1o persistence and diminish pathology in mammals and/or humans.
The invention also provides means, methods and materials for
the generation, manufacturing and use of such recombinant Ad
vectors.
In one embodiment of the invention, the genome of an E1
i5 deleted recombinant Ad vector (as described in W097/00326)
is modified in the E4 promoter region. The entire promoter
region of the E4 transcription unit is deleted and replaced
by a synthetic promoter that consists of an artificial TATA-
box preceded by a specific sequence that is recognized by the
2o DNA binding domain (DBD) of a DBD-transactivating fusion
protein. Said DBD can be derived from a DNA binding protein
that originates from prokaryotes and/or lower eukaryotes but
not from mammals and/or humans. Alternatively, the DBD can be
rationally designed to recognize a synthetic promoter.
2s Transcription of the E4 region of such recombinant Ad genome
would not occur in mammalian and/or human cells. As a
consequence, such an E1 deleted /E4 disabled recombinant Ad
vector does not produce E1 encoded polypeptides and produces
markedly reduced levels of the E4 encoded polypeptides. In
so addition, such a vector produces reduced levels of the viral
late proteins, since it has been shown that E4 polypeptides
are required for the expression of late genes from E1 deleted
vectors (Lusky et al., 1998).
In a more preferred embodiment, the genome of an E1
35 deleted recombinant Ad vector is further deleted for E2A and
modified in the E4 promoter region. The entire promoter
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region of E4 is deleted from the E1+E2A deleted recombinant
Ad DNA and replaced by a synthetic promoter as described
above. Transcription of E4 from such a recombinant Ad genome
would not occur in mammalian and/or human cells. As a
s consequence, such an E1+E2A deleted and E4 disabled Ad vector
does neither produce E1 encoded polypeptides, nor the E2A
encoded polypeptide and produces markedly reduced levels of
the E4 encoded polypeptides. In addition, such a vector
expresses reduced levels of viral late genes, since removing
to E2A and/or disabling E4 functions inhibits the Ad late gene
expression from recombinant Ad vectors (see Figure 1A and
Lusky et al., 1998).
In an even more preferred embodiment, the genome of an
E1+E2A deleted recombinant Ad vector is deleted for the E4
i5 promoter region and modified in the E2 promoter region. The
entire promoter region of the E4 transcription unit is
removed from the E1+E2A deleted recombinant Ad DNA and
replaced by a synthetic promoter as described above. In
addition, the E2 promoter of said vector is inactivated. This
2o inactivation is accomplished by deletion of E2F binding sites
from the E2-early promoter, and/or mutation of the TATA box
and/or CAP site of the E2-early promoter and/or mutation of
the SP1 binding sites and/or TATA-box of the E2-late
promoter. A synthetic promoter, as described above, is
2s inserted directly adjacent to the pTP and pol coding
sequences of the E2B region. Transcription of the E4 and the
E2B region of such recombinant Ad genome would not occur in
mammalian and/or human cells. As a consequence, cells
infected with said El+E2A deleted and E2B+E4 attenuated
3o recombinant Ad vector do neither produce E1 encoded
polypeptides, nor the E2A encoded polypeptide, and produce
markedly reduced levels of the E2B encoded polypeptides and
the E4 encoded polypeptides. In addition, cells infected with
these vectors produce reduced levels of viral late gene
35 products since the expression of late genes is abrogated in
the absence of E2A and E4 encoded proteins.
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In another embodiment, the genome of an E1/E2A deleted
recombinant Ad vector is modified only in the E2 promoter
region, as described above. Transcription of the E2B region
of such recombinant Ad genome would not occur in mammalian
and/or human cells. Therefore, cells infected with such
E1/E2A deleted and E2B attenuated Ad vector do neither
produce E1-encoded polypeptides, nor the E2A encoded
polypeptide, and produce markedly reduced levels of the E2B
encoded polypeptides. In addition, cells infected with such
io vector do not produce viral late encoded proteins since the
expression of viral late gene is abrogated in the absence of
the E2A encoded protein.
In a preferred embodiment, the genome of an E1/E2A
deleted and E2B attenuated recombinant Ad vector is further
i5 deleted for the entire E4 region, or parts thereof. Cells
infected with such E1/E2A/E4 deleted and E2B attenuated Ad
vector do neither produce E1-encoded polypeptides nor the
E2A-encoded polypeptide nor the E4-encoded polypeptides, and
produce markedly reduced levels of the E2B-encoded
2o polypeptides. In addition, said infected cells produce
reduced levels of viral late gene encoded proteins since
expression of viral late genes is abrogated in the absence of
E2A- and/or E4-encoded proteins.
In another embodiment, all the above-described vectors
25 are deleted for the E3 region of the Ad genome, or parts
thereof.
A prerequisite for producing Ad vectors that are deleted
for the coding sequences of essential genes or that are
modified in the promoter region of essential genes is that
3o these defects are complemented in the producing cell lines.
Previously, we have established a cell line that efficiently
complements the deletion of E1 (W097/00326). According to the
invention, this cell line (designated PER. C6) was further
equipped with an expression vector expressing the temperature
35 sensitive mutant of E2A derived from the adenovirus H5ts125,
giving rise to PER.C6/E2A. The cell lines allow the
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manufacturing of high titer batches of recombinant Ad vectors
deleted for E1 or double deleted for E1 and E2A,
respectively. Reversion of the E1 deleted phenotype, i.e. the
formation of replication competent Ad (RCA) in PER.C6 or
s reversion of the E1 + E2A deleted phenotype in PER.C6/E2A was
prevented by elimination of sequence overlap between Ad
sequences present in the cell line and sequences present in
the recombinant vector.
In one embodiment of the invention, the E1 complementing
to cell line PER. C6 is equipped with an expression cassette
coding for a DBD-transactivation fusion protein. Said DBD
recognizes the synthetic sequence preceding the artificial
TATA box in the synthetic promoters) that are present in the
modified genome of the recombinant Ad vectors of the
15 invention. Said DBD is derived from a DNA binding protein
that originates from prokaryotes and/or lower eukaryotes but
not from mammals and/or humans. Alternatively, the DBD is
rationally designed or developed by randomizing techniques to
specifically recognize the synthetic promoter. Because said
2o DBD recognizes the synthetic promoter specifically, it does
not interfere with other transcription processes in PER.C6 or
PER.C6-derived cells and is therefore not toxic. The DBD of
the ectopically expressed DNA binding protein is either fused
to its native transactivation domain or fused to a
2s transcription activation (TA) domain derived from viral
transcription factors (as non-limiting example, the herpes
simplex virus VP16 protein) or fused to a TA domain from
eukaryotic transcription factors (as non-limiting example,
the TA domain of p65-NfkappaB), or fused to a rationally
3o designed TA domain (as non limiting example, an amphipatic a
-helix) or fused to a TA domain that is generated by
randomizing techniques (as non-limiting example, an acidic
blob). Said cell line is suitable for the manufacturing of
recombinant Ad vectors that are deleted for E1 and/or E3 or
35 parts thereof, and in which the E4 promoter region is
replaced by a synthetic promoter according to the invention.
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In a further embodiment, the E1/E2A complementing cell
line PER.C6/E2A is equipped with an expression cassette
coding for the above-described DBD-transactivating fusion
protein. Said cell line is suitable for the manufacturing of
recombinant Ad vectors that are deleted for E1+E2A, and that
either do or do not have a deletion of the entire E3 region
or parts thereof, and in which E4 and/or E2B is equipped with
a synthetic promoter according to the invention.
In yet another embodiment, the E1+E2A complementing cell
1o line PER.C6/E2A is equipped with an expression cassette
coding for the E4-orf6 protein and an expression cassette for
the above described DBD-TA fusion protein. Said cell line is
suitable for the manufacturing of recombinant Ad vectors that
are deleted for E1+E2A+E4 or parts thereof and/or E3 or parts
thereof, and in which the E2 early and late promoters are
knocked-out by mutations and in which E2B is equipped with a
synthetic promoter according to the invention.
In another aspect of the invention, the E4 and/or E2
promoter sequences of the recombinant Ad vector genome are
zo deleted and appropriately replaced by a so-called inducible
promoter, such as, but not limited to, the metallothionein
promoter or the mouse mammary tumor virus (MMTV) promoter.
Hence, the transcription of the E4 and/or the E2B genes can
be switched on or off at will following a specific signal.
z5 Said vectors can be produced in complementing cell lines
expressing E1 and/or E2A and/or E4orf-6 in the presence of
the signal that triggers the expression of the E4 and/or E2
genes.
The vectors according to the invention can be equipped
3o with any foreign nucleic acid, preferably a nucleic acid
encoding a therapeutic molecule. The expression can be driven
by a strong viral enhancer/promoter such as but not limited
to the CMV promoter. More preferably, said expression is
driven by strong mammalian promoters such as but not limited
35 to the EF-la promoter or the UbC promoter to achieve long
term expression of the therapeutic gene.
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The vectors according to the invention can also be
applied for purposes other than gene therapy such as, but not
limited to, functional characterization of gene products in
vitro and in vivo. For instance, the vectors according to the
s invention can be used for overexpression of a variety of
known and novel genes in cell lines, tissues or animals in
order to find genes that encode for proteins with a desired
function such as, but not limited to, those that interfere
with cell proliferation and differentation. For this
io application, it is of critical importance that the vector
itself does not interfere with cellular processes and that it
does not overrule the effect of the transgene. The vectors
according to the invention are replication defective and do
express the remaining viral genes, if at all, only at
is background levels; interference of the vector with the
function and the effect of the gene of interest is therefore
at least in part prevented.
The vectors according to the invention can also be used
as a vaccine. As a non-limiting example, the vectors
2o according to the invention can be equipped with an expression
cassette that codes for a protein against which an immune
response has to be raised. For such application, it is of
critical importance that the vector itself does not
dominantly elicit a response of the immune system but that
2s the immune response, instead, is directed primarily against
the transgene product. Because the vectors according to the
invention are replication defective and express the remaining
viral genes, if at all, only at background levels, it is
expected that the immune response will be directed primarily
3o against the transgene product.
The vectors according to the invention can also be used
for protein production in mammalian cells. Therefore, the
vectors according to the invention can be equipped with an
expression cassette that encodes a protein of interest to be
3s synthesized and processed in said cells. For this
application, it is of importance that the vector itself does
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not interfere with the cellular metabolism which is harmful
to the cell and which impairs synthesis and processing of the
protein of interest. Because the vectors according to the
invention are replication defective and express the remaining
s viral genes, if at all, only at background levels, said .
vectors are less toxic to the cells which, in turn, results
in a prolonged synthesis of the protein of interest.
Adenoviral vectors typically do not or very
inefficiently integrate into the host cell genome. To
1o increase at least in part the integration frequency of
adenovirus vectors elements from integrating viruses can be
included in the vector. The vectors and the gene delivery
vehicles of the invention are suited for integrating vectors
since the vectors are at least in part not toxic to the
i5 cells. The invention therefor provides a recombinant
adenoviral vector and/or a recombinant adenovirus-like gene
delivery vehicle according to the invention, wherein said
adenoviral vector comprises at least one adeno-associated
virus terminal repeat or a functional equivalent thereof.
2o Preferably, said adenovirus vectors comprise at least two
adeno-associated virus terminal repeat or a functional
equivalent thereof. A functional equivalent of an adeno-
associated virus terminal repeat comprises the same
integrating function in kind not necessarily in amount. In a
2s preferred aspect, the adeno-associated virus terminal repeat
is present at the extreme ends of the adenoviral vector.
The invention further provides a recombinant adenoviral
vector and/or a recombinant adenovirus-like gene delivery
vehicle according to the invention comprising elements
3o derived from at least two different adenovirus serotypes.
Such vectors are preferred for a variety of reasons based on
the observation that in this way at least part of the
favourable properties of different adenovirus serotypes may
be combined.
35 The invention further provides a method for ex vivo
production of a gene product in a cell comprising providing
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said cell with a recombinant adenoviral vector and/or a
recombinant adenovirus-like gene delivery vehicle according
to the invention comprising nucleic acid encoding said gene
product, culturing said cell to allow expression of said gene
product and optionally harvesting said cell and/or medium
said cell was exposed to.
In another aspect the invention provides the use of a
recombinant adenoviral vector and/or a recombinant
adenovirus-like gene delivery vehicle according to the
to invention, for the preparation of a medicament.
In yet another aspect the invention provides a vaccine
comprising a recombinant adenoviral vector and/or a
recombinant adenovirus-like gene delivery vehicle according
to the invention, wherein said adenoviral vector comprises a
z5 nucleic acid encoding a proteinaceous molecule against which
an immune response has to be raised.
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EXAMPLES
The invention will now be described with respect to the
generation of recombinant Ad vectors that are deleted for the
5 E1 and/or E2A and/or E4 region, and that, in addition, harbor
E4 and/or E2B genes whose expression is controlled by
synthetic promoters as well as with respect to complementing
cell lines. However, the scope of the present invention is
not intended to be limited thereby.
io
Example 1
Plasmid based system for the generation of recombinant Ad
vectors that are deleted for E1 and/or E2A and in which the
15 E4 and/or E2B promoter is replaced by a synthetic promoter
A. Generation of pBr/Ad.Bam-rITR (ECACC deposit
P97082122)
In order to facilitate blunt end cloning of the ITR
2o sequences, wild-type human adenovirus type 5 (Ad5) DNA was
treated with Klenow enzyme in the presence of excess dNTPs.
After inactivation of the Klenow enzyme and purification by
phenol/chloroform extraction followed by ethanol
precipitation, the DNA was digested with BamHI. This DNA
2s preparation was used without further purification in a
ligation reaction with pBr322 derived vector DNA prepared as
follows: pBr322 DNA was digested with EcoRV and BamHI, de-
phosphorylated by treatment with TSAP enzyme (Life
Technologies) and purified on LMP agarose gel (SeaPlaque
3o GTG). After transformation into competent E.coli DHSa (Life
Techn.) and analysis of ampiciline resistant colonies, one
clone was selected that showed a digestion pattern as
expected for an insert extending from the BamHI site in Ad5
to the right ITR.
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Sequence analysis of the cloning border at the right ITR
revealed that the most 3' G residue of the ITR was missing,
the remainder of the ITR was found to be correct. Said
missing G residue is complemented by the other ITR during
replication.
B. Generation of pBr/Ad.Sal-rITR (ECACC deposit
P97082119)
pBr/Ad.Bam-rITR was digested with BamHI and SalI. The vector
to fragment including the adenovirus insert was isolated from
LMP agarose (SeaPlaque GTG) and ligated to a 4.8 kb SalI-
BamHI fragment obtained from wt Ad5 DNA and purified with the
Geneclean II kit (Bio 101, Inc.). One clone was chosen and
the integrity of the Ad5 sequences was determined by
restriction enzyme analysis. Clone pBr/Ad.Sal-rITR contains
adeno type 5 sequences from the SalI site at by 16746 up to
and including the rITR (missing the most 3' G residue).
C. pBr/Ad. Cla-Bam (ECACC deposi t P97082117)
2o Wild-type Ad type 5 DNA was digested with ClaI and BamHI, and
the 20.6-kb fragment was isolated from gel by electro-
elution. pBr322 was digested with the same enzymes and
purified from agarose gel by Geneclean. Both fragments were
ligated and transformed into competent DHSa. The resulting
clone pBr/Ad.Cla-Bam was analyzed by restriction enzyme
digestion and shown to contain an insert with adenovirus
sequences from by 919 to 21566.
D. Generation of pBr/Ad.AfIII-Bam (ECACC deposit
3o P97082114)
Clone pBr/Ad.Cla-Bam was linearized with EcoRI (in pBr322)
and partially digested with AflII. After heat inactivation of
AflII for 20' at 65°C the fragment ends were filled in with
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27
Klenow enzyme. The DNA was then legated to a blunt double
stranded oligo linker containing a PacI site (5'-
AATTGTCTTAATTAACCGCTTAA-3'). This linker was made by
annealing the following two oligonucleotides: 5'-
AATTGTCTTAATTAACCGC-3' and 5'-AATTGCGGTTAATTAAGAC-3',
followed by blunting with Klenow enzyme. After precipitation
of the legated DNA to change buffer, the legations were
digested with an excess of PacI enzyme to remove concatamers
of the oligo. The 22016 by partial fragment containing Ad5
1o sequences from by 3534 up to 21566 and the vector sequences,
was isolated in LMP agarose (SeaPlaque GTG), re-legated and
transformed into competent DHSa. One clone that was found to
contain the PacI site and that had retained the large adeno
fragment was selected and sequenced at the 5' end to verify
correct insertion of the PacI linker in the (lost) AflII
site.
E. Genera Lion of pBr/Ad . Bam-rITRpac#2 (ECACC depose t
P97082120) and pBr/Ad.Bam-rITR#8 (ECACC deposit P97082121)
2o To allow insertion of a PacI site near the ITR of Ad5 in
clone pBr/Ad.Bam-rITR about 190 nucleotides were removed
between the ClaI site in the pBr322 backbone and the start of
the ITR sequences. This was done as follows: pBr/Ad.Bam-rITR
was digested with ClaI and treated with nuclease Ba131 for
varying lengths of time (2, 5, 10 and 15 minutes). The extend
of nucleotide removal was followed by separate reactions on
pBr322 DNA (also digested at the ClaI site), using identical
buffers and conditions. Ba131 enzyme was inactivated by
incubation at 75°C for 10 minutes, the DNA was precipitated
3o and re-suspended in a smaller volume TE buffer. To ensure
blunt ends, DNA's were further treated with T4 DNA polymerase
in the presence of excess dNTPs. After digestion of the
(control) pBr322 DNA with SalI, satisfactory degradation
0150 bp) was observed in the samples treated for 10 or 15
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28
minutes. The pBr/Ad.Bam-rITR samples that were treated for 10
or 15 minutes were then legated to the above described
blunted PacI linkers (See pBr/Ad.AfIII-Bam). Legations were
purified by precipitation, digested with excess PacI and
s separated from the linkers in an LMP agarose gel. After re-
legation, DNA's were transformed into competent DHSa and
colonies analyzed. Ten clones were selected that showed a
deletion of approximately the desired length and these were
further analyzed by T-track sequencing (T7 sequencing kit,
1o Pharmacia Biotech). Two clones were found with the PacI
linker inserted just downstream of the rITR. After digestion
with PacI, clone #2 has 28 by and clone #8 has 27 by attached
to the ITR.
15 F. Generation of pWE/Ad.AfIII-rITR (ECACC deposit
P97082116)
Cosmid vector pWEl5 (Clontech) was used to clone larger Ad5
inserts. First, a linker containing a unique PacI site was
inserted in the EcoRI sites of pWElS creating pWEl5.pac. To
2o this end, the double stranded PacI oligo as described for
pBr/Ad.AfIII-BamHI was used but now with its EcoRI protruding
ends. The following fragments were then isolated by electro-
elution from agarose gel: pWEl5.pac digested with PacI,
pBr/AflII-Bam digested with PacI and BamHI and pBr/Ad.Bam-
2s rITR#2 digested with BamHI and PacI. These fragments were
legated together and packaged using ~, phage packaging
extracts (Stratagene) according to the manufacturer's
protocol. After infection of host bacteria, colonies were
grown on plates and analyzed for presence of the complete
3o insert. pWE/Ad.AflII-rITR contains all adenovirus type 5
sequences from by 3534 (AflII site) up to and including the
right ITR (missing the most 3' G residue).
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G. Generation of pWE/Ad.AfIII-EcoRI
pWEl5.pac was digested with ClaI and 5' protruding ends were
filled using Klenow enzyme. The DNA was then digested with
PacI and isolated from agarose gel. pWE/AflII-rITR was
s digested with EcoRI and after treatment with Klenow enzyme
digested with PacI. The large 24-kb fragment containing the
adenoviral sequences was isolated from agarose gel and
ligated to the ClaI-digested and blunted pWEl5.pac vector
using the Ligation Expresst'" kit from Clontech. After
transformation of Ultra-competent XL10-Gold cells
(Stratagene), clones were identified that contained the
expected insert. pWE/AflII-EcoRI contains Ad5 sequences from
by 3534-27336.
H. Generation of pBR/Ad.AfIII-Bam.tet0-E2B
First, the shuttle vector pAAO-E-TATA was constructed using
the following primers:
TATAplus: 5'-AGC TTT CTT ATA AAT TTT CAG TGT TAG ACT AGT AAA
TTG CTT AAG-3'
-TATAmin: 5'-AGC TCT TAA GCA ATT TAC TAG TCT AAC ACT GAA AAT
TTA TAA GAA
(The underlined sequences form a modified TATA box.)
The primers TATAplus and TATAmin were annealed to yield a
double stranded DNA fragment flanked by 5' overhangs that are
z5 compatible for ligation with HindIII digested DNA. Thus, the
product of the annealing reaction was used in a ligation
reaction with HindIII digested pGL3-Enhancer Vector (Promega)
to yield pAAO-E-TATA.
Next, the heptamerized tet-operator sequence was
3o amplified from the plasmid pUHC-13-3 (Gossen and Bujard,
1992) in a PCR reaction using the Expand PCR system
(Boehringer) according to the manufacturers protocol. The
following primers were used:
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tet3: 5'-CCG GAG CTC CAT GGC CTA ACT CGA GTT TAC CAC TCC
C-3'
tet5: 5'-CCC AAG CTT AGC TCG ACT TTC ACT TTT CTC-3'
The amplified fragment was digested with SstI and HindIII
5 (SstI and HindIII sites generated in the tet3 and tet5
primers are represented by the nucleotides in bold) and
cloned into SstI/HindIII digested pAAO-E-TATA giving rise to
pAAO-E-TATA-7xtet0. Sequence analysis confirmed the integrity
of the heptamerized tet-operator sequence in the latter
1o plasmid.
Next, pAAO-E-TATA-7xtet0 was digested with NcoI, and the
resulting fragment containing the 7xtet0 sequence was
purified from agarose and used in a ligation reaction with
NcoI digested pNEB.PmAs yielding pNEB.PmAs7xtet0. The plasmid
i5 pNEB.PmAs was obtained as follows: pBR/Ad.AfIII-Bam (ECACC
deposit P97082114) was digested with PmeI/AscI and the
resulting PmeI/AscI fragment containing the 5' end of the E2B
region was purified from agarose and subcloned into PmeI/AscI
digested pNEB 193 (New England BioLabs Inc.) yielding
2o pNEB.PmAs. Then, pNEB.PmAs7xtet0 was digested with AscI, PmeI
and ScaI and the resulting AscI/PmeI fragment (2808 bp)
containing the 5' E2B region preceded by 7xtet0 was purified
from agarose using the Geneclean II kit, and used in a
ligation reaction with PmeI/AscI digested pBR/Ad.AfIII-Bam
2s (the fragment that lacks the 5' end of the E2B region)
yielding pBR/Ad.AflII-Bam.tet0-E2B.
I. Generation of pBR/Ad.Bam-rITRdE2A
Deletion of the E2A coding sequences from pBR/Ad.Bam-rITR
30 (ECACC deposit P97082122) has been accomplished as follows.
The adenoviral sequences flanking the E2A coding region at
the left and the right site were amplified from the plasmid
pBr/Ad.Sal-rITR (ECACC deposit P97082119) in a PCR reaction
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with the Expand PCR system (Boehringer) according to the
manufacturers protocol. The following primers were used:
Ric,~ht flanking sequences (corresponding Ad5 nucleotides
24033 to 25180)
DE2A.SnaBI: 5'-GGC GTA CGT AGC CCT GTC GAA AG-3'
~E2A.DBP-start: 5'-CCA ATG CAT TCG AAG TAC TTC CTT
CTC CTA TAG GC-3'
The amplified DNA fragment was digested with SnaBI and NsiI
(NsiI site is generated in the primer DE2A.DBP-start,
to underlined) .
Left flanking sequences (corresponding Ad5 nucleotides
21557 to 22442):
DE2A.DBP-stop: 5'-CCA ATG CAT ACG GCG CAG ACG G-3'
DE2A.Bam~iI: 5'-GAG GTG GAT CCC ATG GAC GAG-3'
The amplified DNA was digested with BamHI and NsiI (NsiI site
is generated in the primer ~E2A.DBP-stop, underlined).
Subsequently, the digested DNA fragments were ligated into
SnaBI/BamHI digested pBr/Ad.Bam-rITR, yielding pBr/Ad.Bam-
rITR0E2A. The unique NsiI site can be used to introduce an
2o expression cassette for a gene to be transduced by the
recombinant vector.
J. Generation of pBR/Ad.Bam-rITRdE2AdE2p
First, pBR/Ad.Bam-rITR~E2A was digested with AscI and XbaI
2s and the resulting AscI/XbaI fragment containing the E2
promoter region was purified from agarose using the Geneclean
II kit and subcloned into AscI/XbaI digested pNEB 193 (New
England BioLabs Inc.) yielding pNEB-AX. The latter plasmid
contains the Ad E2 early and late promoter sequences. A part
30 of the E2 early promoter sequence was amplified from pNEB-AX
using the primers E2eSpeI and E2eSrf whereas a part of the E2
late promoter sequence was amplified from pNEB-AX using the
primers E2lAat and E2lSpeI.
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E2eSpeI: 5' GGA CTA GTC TAA GTC TTC TCC AGC GGC CAC ACC CGG
3'
E2eSrf: 5' GAG TTA TAC CCT GCC CGG GCG ACC GCA CC 3'
E2lAat: 5' GGG CTG TGG ACG TCG GCT TAC CTT CGC AAG TTC GTA
CCT GAG GAC TAC CAT GCA CAC GAG ATT AGG 3'
E2lSpeI: 5' GCG AAA CTA GTC CTT CAG AGT CAG CGC GCA GTA CTT
GCT AAA AAG AGC CTC CGC 3'
io The nucleotides indicated in bold generate the unique SpeI,
SrfI and AatI restriction sites whereas the underlined
nucleotides generate mutations in the promoter sequences that
knock out the E2F binding sites and mutate the TATA box and
CAP site of the E2 early promoter, and mutate the SP1 binding
sites as well as the TATA box of the E2 late promoter.
The amplification products of the E2 early and late
promoter regions were purified from gel using the Geneclean
II kit and digested with SpeI. Thereafter, the SpeI digested
PCR products were ligated. After chloroform/phenol
2o extraction, the ligated DNA's were digested with AatII and
SrfI. The resulting DNA's were separated in an agarose gel
and the ligation product that comprised the reconstituted and
modified E2 (early and late) promoter was purified from
agarose using the Geneclean II kit. The purified fragment was
2s then used in a ligation reaction with AatII/SrfI digested
pNEB-AX yielding pNEB-AXOE2p. The primers for amplification
and mutation of the E2 promoter were chosen such that part of
the native E2 early promoter spanning the E2F sites and a
putative TATA-box was excluded from amplification. As a
3o result, the modified E2 promoter region that is reconstituted
by ligation of the two PCR products is deleted for the above
mentioned promoter sequences. Next, pNEB-AXOE2p was digested
with AscI and Srfl and the resulting AscI/SrfI fragment
containing the mutated E2 promoter was purified from agarose
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using the Geneclean Spin kit and cloned into Ascl/SrfI
digested pBR/Ad.Bam-rITR0E2A (the fragment that lacks the E2
promoter) giving rise to pBR/Ad.Bam-rITR0E2ADE2p.
K. Generation of pBR/Ad.Bam-rITR.tetO-E4, pBR/Ad.Bam-rITR.d
E2A.tetO-E4 and pBR/Ad.Bam-rITR.dE2A.dE2p.tet0-E4
First, pBR/Ad.Bam-rITR was digested with PacI and Sse83871
and the resulting PacI/Sse83871 fragment containing the E4
region was purified from agarose using the Geneclean II kit
1o and subcloned into PacI/Sse83871 digested pNEB 193 (New
England BioLabs Inc.) giving rise to pNEB-PaSe. The latter
plasmid was used to amplify the Ad sequences that flank the
E4 promoter. Sequences upstream of the E4 promoter were
amplified using the primers 3ITR and 5ITR whereas sequences
downstream of the E4 promoter were amplified using the
primers H3DE4 and AvDE4.
3ITR: 5' CCG GAT CCT TAA TTA AGT TAA CAT CAT C 3'
5ITR: ,5' GAT CCG GAG CTC TAC GTC ACC CGC CCC G 3'
2o H3DE4: 5' CCC AAG CTT AGT CCT ATA TAT ACT CGC TC 3'
AvDE4: 5' CTC CTG CCT AGG CAA AAT AGC 3'
The nucleotides indicated in bold form unique restriction
sites for PacI, SstI, HindIII and AvrII, respectively. The
PCR products were first purified using the QIAquick PCR
z5 purification kit. The PCR product of the sequence downstream
of the E4 promoter was digested with HindIII and AvrII. The
PCR product of the sequence upstream of the E4 promoter was
digested with SstI and ligated to a fragment that contained
the 7xtet0 sequence which was obtained by digestion of pAAO-
3o E-TATA-7xtet0 with SstI and HindIII. The ligation products
were thereafter digested with HindIII and PacI and the
ligation product comprising the region upstream of the E4
promoter linked to the 7xtet0 sequence was purified from gel.
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The latter fragment was used in a ligation reaction with
HindIII/AvrII digested PCR product of the sequence downstream
of the E4 promoter and with PacI/AvrII digested pNEB-PaSe
giving rise to pNEB-PaSe.tet0.
s Next, pNEB-PaSe.tet0 was digested with PacI and Sse83871
and the fragment containing the artificial 7xtet0 promoter
sequences in front of the E4 region was purified from agarose
and cloned into PacI/Sse83871 digested pBR/Ad.Bam-rITR0E2A
giving rise to pBR/Ad.Bam-rITR0E2A.tetO-E4. The latter
to plasmid was used for digestion with PacI and NotI and the
resulting NotI/PacI fragment containing the 7xtet0 promoter
sequences was purified from agarose and used in a ligation
reaction with NotI/PacI digested pBR/Ad.Bam-rITR (the
fragment that lacks the E4 promoter) giving rise to
1s pBR/Ad.Bam-rITRtetO-E4. Finally, the above described
NotI/PacI fragment from pBR/Ad.Bam-rITR.~E2A.tetO-E4 was also
cloned into NotI/PacI digested pBR/Ad.Bam-rITR.~E2A.DE2p (the
fragment that lacks the E4 promoter region) giving rise to
pBR/Ad.Bam-rITR.DE2A.DE2p.tet0-E4.
L. Generation of pWE/Ad.AfIII-rITR.dE2A.tetO-E2B,
pWE/Ad.AfIII-rITR.tetO-E4, pWE/Ad.AfIII-rITR.dE2A,
pWE/Ad.AfIII-rITR.dE2A.tetO-E4, and pWE/Ad.AfIII-rITR.d
E2A.tetO-E2B.tetO-E4.
2s The cosmid pWEl5.pac was linearized by digestion with PacI
and used in a ligation reaction with BamHI/PacI digested
pBR/Ad.AfIII-Bam.tet0-E2B and BamHI/PacI digested pBR/Ad.Bam-
rITR.DE2A.DE2p. The ligation mixture was used in a packaging
reaction using 7~ phage-packaging extracts (Stratagene)
3o according to the manufacturer's protocol, yielding the cosmid
pWE/Ad.AfIII-rITR.DE2A.tetO-E2B.
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Similarly, PacI-linearized pWEl5.pac was used in a
ligation reaction with BamHI/PacI digested pBR/Ad.AfIII-Bam
and BamHI/PacI digested pBR/Ad.Bam-rITR-tet0-E4. The ligation
mixture was used in a packaging reaction using ~, phage-
5 packaging extracts (Stratagene) according to the
manufacturer's protocol, yielding the cosmid pWE/Ad.AfIII-
rITR.tetO-E4.
Similarly, PacI-linearized pWEl5.pac was used in a
ligation reaction with BamHI/PacI digested pBR/Ad.AfIII-Bam
1o and BamHI/PacI digested pBR/Ad.Bam-rITR.~E2A. The ligation
mixture was used in a packaging reaction using ~, phage-
packaging extracts (Stratagene) according to the
manufacturer's protocol, yielding the cosmid pWE/Ad.AfIII-
rITR.DE2A.
15 Similarly, PacI-linearized pWEl5.pac was used in a
ligation reaction with BamHI/PacI digested pBR/Ad.AfIII-Bam
and BamHI/PacI digested pBR/Ad.Bam-rITR.DE2A.tetO-E4. The
ligation mixture was used in a packaging reaction using ~,
phage-packaging extracts (Stratagene) according to the
2o manufacturer's protocol, yielding the cosmid pWE/Ad.AfIII-
rITR.~E2A.tetO-E4.
Similarly, PacI-linearized pWEl5.pac was used in a
ligation reaction with BamHI/PacI digested pBR/Ad.AfIII-
Bam.tet0-E2B and BamHI/PacI digested pBR/Ad.Bam-rITR.DE2A.0
z5 E2p.tet0-E4. The ligation mixture was used in a packaging
reaction using 7~ phage-packaging extracts (Stratagene)
according to the manufacturer's protocol, yielding the cosmid
pWE/Ad.AfIII-rITR.~E2A.tetO-E2B.tetO-E4.
3o M. Generation of the adapter plasmids
Adapter plasmid pMLP.TK (described in EP 95202213) was
modified as follows: SV40 polyA sequences were amplified with
primer SV40-1 (introduces a BamHI site) and SV40-2
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(introduces a BglII site). In addition, Ad5 sequences present
in this construct (from nt. 2496 to nt. 2779; Ad5 sequences
nt. 3511 to 3794) were amplified with primers Ad5-1
(introduces a BglII site) and Ad5-2.
SV40-1: 5'-GGGGGATCCGAACTTGTTTATTGCAGC-3'
SV40-2: 5'-GGGAGATCTAGACATGATAAGATAC-3'
Ad5-1: 5'-GGGAGATCTGTACTGAAATGTGTGGGC-3'
Ad5-2: 5'-GGAGGCTGCAGTCTCCAACGGCGT-3'
to
Both PCR fragments were digested with BglII and ligated. The
ligation product was amplified with primers SV40-1 and Ad5-2
and digested with BamHI and AflII. The digested fragment was
then ligated into pMLP.TK predigested with the same enzymes.
The resulting construct, named pMLPI.TK, contains a deletion
in adenovirus E1 sequences from nt. 459 to nt. 3510.
This plasmid was used as the starting material to make a
new vector in which nucleic acid molecules comprising
specific promoter and gene sequences can be easily exchanged.
2o First, a PCR fragment was generated from pZipOMo+PyF101(N-)
template DNA (described in PCT/NL96/00195) with the following
primers:
LTR-1: 5'-CTG TAC GTA CCA GTG CAC TGG CCT AGG CAT GGA AAA ATA
CAT AAC TG-3' and
LTR-2: 5'-GCG GAT CCT TCG AAC CAT GGT AAG CTT GGT ACC GCT AGC
GTT AAC CGG GCG ACT CAG TCA ATC G-3'.
Pwo DNA polymerase (Boehringer Mannheim) was used according
to manufacturers protocol with the following temperature
cycles: once 5 minutes at 95°C; 3 minutes at 55°C; and 1
3o minute at 72°C, and 30 cycles of 1 minute at 95°C, 1 minute
at 60°C, 1 minute at 72°C, followed by once 10 minutes at
72°
C. The PCR product was then digested with BamHI and ligated
into pMLPlO (Levrero et al., 1991) digested with PvuII and
BamHI, thereby generating vector pLTRlO. This vector contains
s5 adenoviral sequences from by 1 up to by 454 followed by a
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promoter consisting of a part of the Mo-MuLV LTR having its
wild-type enhancer sequences replaced by the enhancer from a
mutant polyoma virus (PyF101). The promoter fragment was
designated L420. Sequencing confirmed correct amplification
s of the LTR fragment however the most 5' bases in the pcr
fragment were missing so that the PvuII site was not
restored. Next, the coding region of the murine HSA gene was
inserted. pLTRlO was digested with BstBI followed by Klenow
treatment and digestion with NcoI. The HSA gene was obtained
1o by PCR amplification on pUCl8-HSA (Kay et al., 1990) using
the following primers:
HSA1, 5'-GCG CCA CCA TGG GCA GAG CGA TGG TGG C-3' and
HSA2, 5'-GTT AGA TCT AAG CTT GTC GAC ATC GAT CTA CTA ACA GTA
GAG ATG TAG AA-3'.
15 The 269 bp-amplified fragment was sub-cloned in a shuttle
vector using the NcoI and BglII sites. Sequencing confirmed
incorporation of the correct coding sequence of the HSA gene,
but with an extra TAG insertion directly following the TAG
stop codon. The coding region of the HSA gene, including the
2o TAG duplication was then excised as a NcoI (sticky)-SalI
(blunt) fragment and cloned into the 3.5 kb NcoI
(sticky)/BstBI (blunt) fragment from pLTRlO, resulting in
pLTR-HSA10.
Finally, pLTR-HSA10 was digested with EcoRI and BamHI after
2s which the fragment containing the left ITR, packaging signal,
L420 promoter and HSA gene was inserted into vector pMLPI.TK
digested with the same enzymes and thereby replacing the
promoter and gene sequences. This resulted in the new adapter
plasmid pAdS/L420-HSA that contains convenient recognition
3o sites for various restriction enzymes around the promoter and
gene sequences. SnaBI and AvrII can be combined with HpaI,
NheI, KpnI, HindIII to exchange promoter sequences, while the
latter sites can be combined with the ClaI or BamHI sites 3'
from HSA coding region to replace genes in this construct.
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The vector pAdS/L420-HSA was then modified to create a SalI
or PacI site upstream of the left ITR. Hereto pAdS/L420-HSA
was digested with EcoRI and legated to a PacI linker (5'-
AATTGTCTTAATTAACCGCTTAA-3'). The legation mixture was
digested with PacI and relegated after isolation of the
linear DNA from agarose gel to remove concatamerised linkers.
This resulted in adapter plasmid pAdS/L420-HSA.pac.
Another adapter plasmid that was designed to allow easy
exchange of nucleic acid molecules was made by replacing the
1o promoter, gene and polyA sequences in pAdS/L420-HSA with the
CMV promoter, a multiple cloning site, an intron and a polyA
signal. For this purpose, pAd/L420-HSA was digested with
AvrII and BglII followed by treatment with Klenow to obtain
blunt ends. The 5.1 kb fragment with pBr322 vector and
i5 adenoviral sequences was isolated. and legated to a blunt 1570
by fragment from pcDNAl/amp (Invitrogen) obtained by
digestion with HhaI and AvrII followed by treatment with T4
DNA polymerase. This adapter plasmid was named pAdS/Clip. To
enable removal of vector sequences from the left ITR in
2o pAdS/Clip, this plasmid was partially digested with EcoRI and
the linear fragment was isolated. An oligo of the sequence
5' TTAAGTCGAC-3' was annealed to itself resulting in a linker
with a SalI site and EcoRI overhang. The linker was legated
to the partially digested pAdS/Clip vector and clones were
2s selected that had the linker inserted in the EcoRI site 23 by
upstream of the left adenovirus ITR in pAdS/Clip resulting in
pAdS/Clip.sal.
To create an adapter plasmid that only contains a
polylinker sequence and no promoter or polyA sequences,
3o pAdS/L420-HSA.pac was digested with AvrII and BglII. The
vector fragment was legated to a linker oligonucleotide
digested with the same restriction enzymes. The linker was
made by annealing oligos of the following sequence:
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PLL-1: 5'- GCC ATC CCT AGG AAG CTT GGT ACC GGT GAA TTC GCT
AGC GTT AAC GGA TCC TCT AGA CGA GAT CTG G-3' and
PLL-2: 5'- CCA GAT CTC GTC TAG AGG ATC CGT TAA CGC TAG CGA
ATT CAC CGG TAC CAA GCT TCC TAG GGA TGG C-3'.
The annealed linkers were digested with AvrII and BglII and
separated from small ends by column purification (Qiaquick
nucleotide removal kit) according to manufacturers
recommendations. The linker was then ligated to the
AvrII/BglII digested pAdS/L420-HSApac fragment. A clone,
io named pAdMire, was selected that had the linker incorporated
and was sequenced to check the integrity of the insert.
Adapter plasmid pAdMire enables easy insertion of complete
expression cassettes.
An adapter plasmid containing the human CMV promoter
that mediates high expression levels in human cells was
constructed as follows: pAdS/L420-HSA.pac was digested with
AvrII and 5' protruding ends were filled in using Klenow
enzyme. A second digestion with HindIII resulted in removal
of the L420 promoter sequences. The vector fragment was
2o isolated and ligated to a PCR fragment containing the CMV
promoter sequence. This PCR fragment was obtained after
amplification of CMV sequences from pCMVLacI (Stratagene)
with the following primers:
CMVplus: 5'-GATCGGTACCACTGCAGTGGTCAATATTGGCCATTAGCC-3' and
CMVminA: 5'-GATCAAGCTTCCAATGCACCGTTCCCGGC-3'.
The PCR fragment was first digested with PstI (underlined in
CMVplus) after which the 3'-protruding ends were removed by
treatment with T4 DNA polymerase. Then the DNA was digested
with HindIII (underlined in CMVminA) and ligated into the
3o above described pAdS/L420-HAS.pac vector fragment digested
with AvrII and HindIII. The resulting plasmid was named
pAdS/CMV-HSApac. This plasmid was then digested with HindIII
and BamHI and the vector fragment was isolated and ligated to
the polylinker sequence obtained after digestion of pAdMire
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with HindIII and BglII. The resulting plasmid was named
pAdApt. Adapter plasmid pAdApt contains nucleotides -735 to
+95 of the human CMV promoter (Boshart et al., 1985).
The adapter plasmid pCMV.LacZ was generated as
5 follows: The plasmid pCMV.TK (EP 95-202 213) was digested
with HindIII, blunted with Klenow and dNTPs and subsequently
digested with SalI. The DNA fragment containing the CMV
promoter was isolated. The plasmid pMLP.nlsLacZ (EP 95-202
213) was digested with KpnI, blunted with T4 DNA polymerase
io and subsequently digested with SalI. The DNA fragment
containing the LacZ gene and adjacent adenoviral sequences
was isolated. Next, the two DNA fragments were ligated with
T4 DNA ligase in the presence of ATP, giving rise to
pCMV.nlsLacZ.
15 The adapter plasmid pAdS/CLIP.LacZ was generated as
follows: The E.coli LacZ gene was amplified from the plasmid
pMLP.nlsLacZ (EP 95-202 213) by PCR with the primers
5'-GGGGTGGCCAGGGTACCTCTAGGCTTTTGCAA-3' and
5'-GGGGGGATCCATAAACAAGTTCAGAATCC-3'.
20 The PCR reaction was performed using Ex Taq (Takara)
according to the suppliers protocol at the following
amplification program: 5 minutes 94°C, 1 cycle; 45 seconds
94°C and 30 seconds 60°C and 2 minutes 72°C, 5 cycles; 45
seconds 94°C and 30 seconds 65°C and 2 minutes 72°C, 25
25 cycles; 10 minutes 72; 45 seconds 94°C and 30 seconds 60°C
and 2 minutes 72°C, 5 cycles, I cycle. The PCR product was
subsequently digested with Kpnl and BamHI and the digested
DNA fragment was ligated into KpnI/BamHI digested pcDNA3
(Invitrogen), giving rise to pcDNA3.nlsLacZ. Next, the
3o plasmid pAd/CLIP was digested with SpeI. The large fragment
containing part of the 5' part CMV promoter and the
adenoviral sequences was isolated. The plasmid pcDNA3.nlsLacZ
was digested with SpeI and the fragment containing the 3'part
of the CMV promoter and the lacZ gene was isolated.
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Subsequently, the fragments were ligated, giving rise to
pAd/CLIP.LacZ. The reconstitution of the CMV promoter was
confirmed by restriction digestion. Next the LacZ gene was
digested from pAd/CLIP.LacZ with KpnI and XhaI and isolated
s from agarose gel. The plasmid pAdApt was digested with KpnI
and X~baI and the large fragment was purified from agarose
gel. Next, the LacZ fragment and pAdApt fragment were
ligated, yielding pAdApt.LacZ.
The adapter plasmid pAdS/CLIP.Luc was generated as
to follows: The plasmid pCMV.Luc (EP 95-202 213) was digested
with HindIII and BamHI. The DNA fragment containing the
luciferase gene was isolated. The adapter plasmid pAd/CLIP
was digested with HindIII and BamHI, and the large fragment
was isolated. Next, the isolated DNA fragments were ligated,
i5 giving rise to pAdS/CLIP.Luc.
The adapter plasmid pAdS/ULIP.LacZ.sal was
generated as follows: First, the Ubiquitin C promoter (Nenoi
et al., 1996) was amplified from genomic DNA from human
osteosarcoma cells (U2-OS) by a PCR reaction using the
2o following primers:
Upstream: 5'-GAT CGA TAT CAC GGC GAG CGC TGC CAC G-3'
Downstream: 5'-GAT CGA TAT CTG TCT AAC AAA AAA GCC
AAA AAC GGC C-3'
(The underlined sequences form EcoRV restriction sites).
25 Next, pAd/CLIP.Sal was digested with SpeI to remove the
CMV promoter sequences and the remaining part of the vector
was religated to yield pAd/LIP.Sal. The latter plasmid was
digested with EcoRV and used in a ligation reaction with
EcoRV digested Ubiquitin C promoter containing PCR product
3o yielding pAdS/ULIP.Sal. Then, pAdS/ULIP.Sal was digested with
NotI, and the resulting overhang was filled in by Klenow
treatment and thereafter digested by XbaI, followed by
dephosphorylation. The resulting DNA was used in a ligation
reaction with KpnI/XbaI digested pAd/CLIP.LacZ.Sal, from
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which the KpnI sites was made blunt by treatment with T4 DNA
polymerase in the absence of nucleotides, yielding
pAd/ULIP.LacZ.Sal.
The adapter plasmid pAd/EF-la.LacZ.Pac is generated as
follows. The EF-la promoter is amplified by PCR from the
plasmid pEF-BOS (Mizushima and Nagata, 1990) using the
primers
Upstream: 5'-GAT CGG TAC CCG TGA GGC TCC GGT GCC C-3'
Downstream: 5'-GAT CGG TAC CAA GCT TTT CAC GAC ACC TGA AA
1o TGG-3' .
(The underlined sequences form Acc65I restriction sites).
Next, pAdApt.LacZ is digested with AvrII and Acc65I, treated
with Klenow enzyme and relegated to generate pAdS/Nop.LacZ.
The latter plasmid is digested with Acc65I and used in a
legation reaction with Acc65I digested PCR product containing
the EF-la promoter, yielding pAdS/EF-la.LacZ.
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Example 2
Generation of producer cell lines for the production of
recombinant adenoviral vectors deleted in early region 1
and early region 2A
Here is described the generation of cell lines for the
production of recombinant adenoviral vectors that are deleted
in early region 1 (E1) and early region 2A (E2A). The
producer cell lines complement for the E1 and E2A deletion
to from recombinant adenoviral vectors in trans by constitutive
expression of both E1 and E2A genes. The pre-established Ad5-
E1 transformed human embryo retinoblast cell line PER.C6 (WO
97/00326) was further equipped with E2A expression cassettes.
The adenoviral E2A gene encodes a 72 kDa DNA Binding
Protein with has a high affinity for single stranded DNA.
Because of its function, constitutive expression of DBP is
toxic for cells. The ts125E2A mutant encodes a DBP which has
a Pro-~Ser substitution of amino acid 413 (van der Vliet,
1975). Due to this mutation, the ts125E2A encoded DBP is
2o fully active at the permissive temperature of 32°C, but does
not bind to ssDNA at the non-permissive temperature of 39°C.
This allows the generation of cell lines that constitutively
express E2A, which is not functional and is not toxic at the
non-permissive temperature of 39°C. Temperature sensitive E2A
2s gradually becomes functional upon temperature decrease and
becomes fully functional at a temperature of 32°C, the
permissive temperature.
A. Generation of plasmids expressing the wild type E2A-
30 or temperature sensitive ts125E2A gene.
pcDNA3wtE2A: The complete wild-type early region 2A
(E2A) coding region was amplified from the plasmid
pBR/Ad.Bam-rITR (ECACC deposit P97082122) with the primers
DBPpcrl and DBPpcr2 using the ExpandT"" Long Template PCR
35 system according to the standard protocol of the supplier
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(Boehringer Mannheim). The PCR was performed on a Biometra
Trio Thermoblock, using the following amplification program:
94°C for 2 minutes, 1 cycle; 94°C for 10 seconds + 51°C
for
30 seconds + 68°C for 2 minutes, 1 cycle; 94°C for 10 seconds
s + 58°C for 30 seconds + 68°C for 2 minutes, 10 cycles;
94°C
for 10 seconds + 58°C for 30 seconds + 68°C for 2 minutes
with 10 seconds extension per cycle, 20 cycles; 68°C for 5
minutes, 1 cycle. The primer DBPpcrl: CGG GAT CCG CCA CCA TGG
CCA GTC GGG AAG AGG AG (5' to 3') contains a unique BamHI
Zo restriction site (underlined) 5' of the Kozak sequence
(italic) and start codon of the E2A coding sequence. The
primer DBPpcr2: CGG AAT TCT TAA AAA TCA AAG GGG TTC TGC CGC
(5' to 3') contains a unique EcoRI restriction site
(underlined) 3' of the stop codon of the E2A coding sequence.
15 The bold characters refer to sequences derived from the E2A
coding region. The PCR fragment was digested with BamHI/EcoRI
and cloned into BamHI/EcoRI digested pcDNA3 (Invitrogen),
giving rise to pcDNA3wtE2A.
pcDNA3tsE2A: The complete ts125E2A-coding region was
2o amplified from DNA isolated from the temperature sensitive
adenovirus mutant H5ts125 (Ensinger et al., 1972; van der
Vliet et al., 1975). The PCR amplification procedure was
identical to that for the amplification of wtE2A. The PCR
fragment was digested with BamHI/EcoRI and cloned into
25 BamHI/EcoRI digested pcDNA3 (Invitrogen), giving rise to
pcDNA3tsE2A. The integrity of the coding sequence of wtE2A
and tsE2A was confirmed by sequencing.
B. Growth characteristics of producer cells for the
3o production of recombinant adenoviral vectors cultured at 32-,
37- and 39°C.
PER. C6 cells were cultured in Dulbecco's Modified Eagle
Medium (DMEM, Gibco BRL) supplemented with 10% Fetal Bovine
Serum (FBS, Gibco BRL) and lOmM MgCl2 in a 10% COz atmosphere
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at either 32°C, 37°C or 39°C. At day 0, a total of 1 x
106
PER. C6 cells were seeded per 25cmz tissue culture flask
(Nunc) and the cells were cultured at either 32°C, 37°C or
39°C. At day 1-8, cells were counted. Figure 2 shows that the
s growth rate and the final cell density of the PER. C6 culture
at 39°C are comparable to that at 37°C. The growth rate and
final density of the PER. C6 culture at 32°C were slightly
reduced as compared to that at 37°C or 39°C. No significant
cell death was observed at any of the incubation
1o temperatures. Thus PER.C6 performs very well both at 32°C and
39°C, the permissive and non-permissive temperature for
ts125E2A, respectively.
C. Transfection of PER. C6 with E2A expression vectors;
s5 colony formation and generation of cell lines
One day prior to transfection, 2 x 106 PER. C6 cells were
seeded per 6 cm tissue culture dish (Greiner) in DMEM,
supplemented with 10% FBS and lOmM MgCl2 and incubated at
37°C in a 10% COzatmosphere. The next day, the cells were
2o transfected with 3, 5 or 8~.g of either pcDNA3, pcDNA3wtE2A or
pcDNA3tsE2A plasmid DNA per dish, using the LipofectAMINE
PLUST~~ Reagent Kit according to the standard protocol of the
supplier (Gibco BRL), except that the cells were transfected
at 39°C in a 10% COZ atmosphere. After the transfection, the
z5 cells were constantly kept at 39°C, the non-permissive
temperature for ts125E2A. Three days later, the cells were
put in DMEM supplemented with 10% FBS, lOmM MgCl2 and
0.25mg/ml 6418 (Gibco BRL), and the first 6418 resistant
colonies appeared at 10 days post transfection. As shown in
3o table 1, there was a dramatic difference between the total
number of colonies obtained after transfection of pcDNA3
("'200 colonies) or pcDNA3tsE2A ("'100 colonies) and
pcDNA3wtE2A (only 4 colonies). These results indicate that
the toxicity of constitutively expressed E2A can be overcome
35 by using a temperature sensitive mutant of E2A (ts125E2A) and
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culturing of the cells at the non-permissive temperature of
39°C.
From each transfection, a number of colonies was
picked by scraping the cells from the dish with a pipette.
s The detached cells were subsequently put into 24 wells tissue
culture dishes (Greiner) and cultured further at 39°C in a
10% COz atmosphere in DMEM, supplemented with 10% FBS, lOmM
MgClz and 0.25mg/ml 6418. As shown in table 1, 100% of the
pcDNA3 transfected colonies (4/4) and 82% of the pcDNA3tsE2A
io transfected colonies (37/45) were established to stable cell
lines (the remaining 8 pcDNA3tsE2A transfected colonies grew
slowly and were discarded). In contrast, only 1 pcDNA3wtE2A-
transfected colony could be established. The other 3 died
directly after picking.
15 Next, the E2A expression levels in the different cell
lines were determined by Western blotting. The cell lines
were seeded on 6 well tissue culture dishes and sub-confluent
cultures were washed twice with PBS (NPBI) and lysed and
scraped in RIPA (1% NP-40, 0.5% sodium deoxycholate and 0.1%
2o SDS in PBS, supplemented with 1mM
phenylmethylsulfonylfluoride and 0.1 mg/ml trypsin
inhibitor). After 15 minutes incubation on ice, the lysates
were cleared by centrifugation. Protein concentrations were
determined by the Bio-Rad protein assay, according to
2s standard procedures of the supplier (BioRad). Equal amounts
of whole-cell extract were fractionated by SDS-PAGE on 10%
gels. Proteins were transferred onto Immobilon-P membranes
(Millipore) and incubated with the aDBP monoclonal antibody
B6 (Reich et al., 1983). The secondary antibody was a
3o horseradish-peroxidase-conjugated goat anti mouse antibody
(BioRad). The Western blotting procedure and incubations were
performed according to the protocol provided by Millipore.
The complexes were visualized with the ECL detection system
according to the manufacturer's protocol (Amersham). Figure 3
35 shows that all of the cell lines derived from the pcDNA3tsE2A
transfection expressed the 72-kDa E2A protein (left panel,
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lanes 4-14; middle panel, lanes 1-13; right panel, lanes 1-
12). In contrast, the only cell line derived from the
pcDNAwtE2A transfection did not express the E2A protein (left
panel, lane 2). No E2A protein was detected in extract from a
cell line derived from the pcDNA3 transfection (left panel,
lane 1), which served as a negative control. Extract from
PER. C6 cells transiently transfected with pcDNA3ts125 (left
panel, lane 3) served as a positive control for the Western
blot procedure. These data confirmed that constitutive
to expression of wtE2A is toxic for cells and that using the
ts125 mutant of E2A could circumvent this toxicity.
D. Complementation of E2A deletion in adenoviral vectors
on PER. C6 cells constituti vely expressing full-length
ts125E2A.
The adenovirus Ad5.d1802 is an Ad 5 derived vector deleted
for the major part of the E2A coding region and does not
produce functional DBP (Rice et al., 1985). Ad5.d1802.was
used to test the E2A trans-complementing activity of PER. C6
2o cells constitutively expressing ts125E2A. Parental PER. C6
cells or PER.C6tsE2A clone 3-9 were cultured in DMEM,
supplemented with 10% FBS and lOmM MgClz at 39°C and 10% COZ
in 25 cm2 flasks and either mock infected or infected with
Ad5.d1802 at an m.o.i. of 5. Subsequently the infected cells
were cultured at 32°C and cells were screened for the
appearance of a cytopathic effect (CPE) as determined by
changes in cell morphology and detachment of the cells from
the flask. Full CPE appeared in the Ad5.d1802 infected
PER.C6tsE2A clone 3-9 within 2 days. No CPE appeared in the
3o Ad5.d1802 infected PER. C6 cells or the mock infected cells.
These data showed that PER. C6 cells constitutively expressing
ts125E2A complemented in trans for the E2A deletion in the
Ad5.d1802 vector at the permissive temperature of 32°C.
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E. Serum-free suspension culture of PER.C6tsE2A cell
lines.
Large-scale production of recombinant adenoviral vectors
for human gene therapy requires an easy and scaleable
culturing method for the producer cell line, preferably a
suspension culture in medium devoid of any human or animal
constituents. To that end, the cell line PER.C6tsE2A c5-9
(designated c5-9) was cultured at 39°C and 10% COZ in a 175
cm2 tissue culture flask (Nunc) in DMEM, supplemented with
l0 10% FBS and lOmM MgCl2. At sub-confluency (70-80% confluent),
the cells were washed with PBS (NPBI) and the medium was
replaced by 25 ml serum free suspension medium Ex-cellTM 525
(JRH) supplemented with 1 x L-Glutamine (Gibco BRL),
hereafter designated SFM. Two days later, cells were detached
from the flask by flicking and the cells were centrifuged at
1,000 rpm for 5 minutes. The cell pellet was resuspended in 5
ml SFM and 0.5 ml cell suspension was transferred to a 80 cm2
tissue culture flask (Nunc), together with 12 ml fresh SFM.
After 2 days, cells were harvested (all cells are in
2o suspension) and counted in a Burker cell counter. Next, cells
were seeded in a 125 ml tissue culture erlenmeyer (Corning)
at a seeding density of 3 x 105 cells per ml in a total
volume of 20 ml SFM. Cells were further cultured at 125 RPM
on an orbital shaker (GFL) at 39°C in a 10% COzatmosphere.
z5 Cells were counted at day 1-6 in a Burker cell counter. In
Figure 4, the mean growth curve from 8 cultures is shown.
PER.C6tsE2A c5-9 performed well in serum free suspension
culture. The maximum cell density of approximately 2 x 106
cells per ml is reached within 5 days of culture.
F. Growth characteristics of PER.C6 and PER.C6/E2A at
37°C and 39°C.
PER. C6 cells or PER.C6ts125E2A (c8-4) cells were
cultured in Dulbecco's Modified Eagle Medium (DMEM, Gibco
BRL) supplemented with 10% Fetal Bovine Serum (FBS, Gibco
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BRL) and lOmM MgClz in a 10% COz atmosphere at either 37°C
(PER.C6) or 39°C (PER.C6ts125E2A c8-4). At day 0, a total of
1 x 106 cells were seeded per 25cm2 tissue culture flask
(Nunc) and the cells were cultured at the respective
s temperatures. At the indicated time points, cells were
counted. The growth of PER.C6 cells at 37°C was comparable to
the growth of PER.C6ts125E2A c8-4 at 39°C (Figure 5). This
shows that constitutive expression of ts125E2A encoded DBP
had no adverse effect on the growth of cells at the non-
lo permissive temperature of 39°C.
G. Stability of PER.C6ts125E2A
For several passages, the PER.C6ts125E2A cell line clone
8-4 was cultured at 39°C and 10% COZ in a 25 cmz tissue
15 culture flask (Nunc) in DMEM, supplemented with 10% FBS and
mM MgCl2in the absence of selection pressure (G418). At
sub-confluency (70-80% confluent), the cells were washed with
PBS (NPBI) and lysed and scraped in RIPA (1% NP-40, 0.5%
sodium deoxycholate and 0.1% SDS in PBS, supplemented with
1mM phenylmethylsulfonylfluoride and 0.1 mg/ml trypsin
inhibitor). After 15 minutes incubation on ice, the lysates
were cleared by centrifugation. Protein concentrations were
determined by the BioRad protein assay, according to standard
procedures of the supplier (BioRad). Equal amounts of whole-
cell extract were fractionated by SDS-PAGE in 10% gels.
Proteins were transferred onto Immobilon-P membranes
(Millipore) and incubated with the aDBP monoclonal antibody
B6 (Reich et al., 1983). The secondary antibody was a
horseradish-peroxidase-conjugated goat anti mouse antibody
(BioRad). The Western blotting procedure and incubations were
performed according to the protocol provided by Millipore.
The complexes were visualized with the ECL detection system
according to the manufacturer's protocol (Amersham). The
expression of ts125E2A encoded DBP was stable for at least 16
passages, which is equivalent to approximately 40 cell
doublings (Figure 6). No decrease in DBP levels was observed
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during this culture period, indicating that the expression of
ts125E2A was stable, even in the absence of 6418 selection
pressure.
s Example 3
Generation of tTA expressing packaging cell lines
A. Generation of a plasmid from which the tTA gene is
expressed.
io pcDNA3.1-tTA: The tTA gene, a fusion of the tetR and
VP16 genes, was removed from the plasmid pUHD 15-1 (Gossen
and Bujard, 1992) by digestion using the restriction enzymes
BamHI and EcoRI. First, pUHDl5-1 was digested with EcoRI. The
linearized plasmid was treated with Klenow enzyme in the
i5 presence of dNTPs to fill in the EcoRI sticky ends. Then, the
plasmid was digested with BamHI. The resulting fragment, 1025
by in length, was purified from agarose. Subsequently, the
fragment was used in a ligation reaction with BamHI/EcoRV
digested pcDNA 3.1 HYGRO (-) (Invitrogen) giving rise to
2o pcDNA3.1-tTA. After transformation into competent E. Coli DH5
a (Life Techn.) and analysis of ampiciline resistant
colonies, one clone was selected that showed a digestion
pattern as expected for pcDNA3.1-tTA.
25 B. Transfection of PER.C6 and PER.C6/E2A with the tTA
expression vector; colony formation and generation of cell
lines
One day prior to transfection, 2x106 PER.C6 or
PER.C6/E2A cells were seeded per 60 mm tissue culture dish
30 (Greiner) in Dulbecco's modified essential medium (DMEM,
Gibco BRL) supplemented with 10% FBS (JRH) and 10 mM MgClz
and incubated at 37°C in a 10% COz atmosphere. The next day,
cells were transfected with 4-8 ~g of pcDNA3.1-tTA plasmid
DNA using the LipofectAMINE PLUST"' Reagent Kit according to
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the standard protocol of the supplier (Gibco BRL). The cells
were incubated with the LipofectAMINE PLUS~'~"'-DNA mixture for
four hours at 37°C and 10% CO2. Then, 2 ml of DMEM
supplemented with 20% FBS and 10 mM MgCl2 was added and cells
were further incubated at 37°C and 10% CO2. The next day,
cells were washed with PBS and incubated in fresh DMEM
supplemented with 10% FBS, 10 mM MgCl2 at either 37°C
(PER.C6) or 39°C (Per.C6/E2A) in a 10% COZ atmosphere for
three days. Then, the media were exchanged for selection
to media; PER. C6 cells were incubated with DMEM supplemented
with 10% FBS, 10 mM MgCl2 and 50 ~g/ml hygromycin B (GIBCO)
while PER.C6/E2A cells were maintained in DMEM supplemented
with 10% FBS, 10 mM MgCl2 and 100 ~g/ml hygromycin B.
Colonies of cells that resisted the selection appeared within
i5 three weeks while nonresistant cells died during this period.
From each transfection, a number of independent,
hygromycin resistant cell colonies were picked by scraping
the cells from the dish with a pipette and put into 2.5 cm2
dishes (Greiner) for further growth in DMEM containing 10%
2o FBS, 10 mM MgCl2 and supplemented with 50 ~,g/ml (PERC.6
cells) or 100 ~g/ml (PERC.6/E2A cells) hygromycin in a 10%
COz atmosphere and at 37°C or 39°C, respectively.
Next, it was determined whether these hygromycin-
resistant cell colonies expressed functional tTA protein.
25 Therefore, cultures of PER.C6/tTA or PER/E2A/tTA cells were
transfected with the plasmid pUHC 13-3 that contains the
reporter gene luciferase under the control of the 7xtet0
promoter (Gossens and Bujard, 1992). To demonstrate that the
expression of luciferase was mediated by tTA, one half of the
3o cultures was maintained in medium without doxycycline. The
other half was maintained in medium with 8 ~,g/ml doxycycline
(Sigma). The latter drug is an analogue of tetracycline and
binds to tTA and inhibits its activity. All PER.C6/tTA and
PER/E2A/tTA cell lines yielded high levels of luciferase,
35 indicating that all cell lines expressed the tTA protein
(Figure 7). In addition, the expression of luciferase was
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greatly suppressed when the cells were treated with
doxycycline. Collectively, the data showed that the isolated
and established hygromycin-resistant PER. C6 and PER/E2A cell
clones all expressed functional tTA.
Example 4
Generation of recombinant adenoviral vectors.
A. E1-deleted recombinant adenoviruses with wt E3 sequences
to To generate E1 deleted recombinant adenoviruses with the
plasmid-based system, the following constructs are prepared:
a) An adapter construct containing the expression cassette
with the gene of interest linearized with a restriction
enzyme that cuts at the 3' side of the overlapping
adenoviral genome fragment, preferably not containing any
pBr322 vector sequences, and
b) A complementing adenoviral genome construct pWE/Ad.AfIII-
rITR (ECACC deposit P97082116) digested with PacI.
These two DNA molecules are further purified by
2o phenol/chloroform extraction and ethanol precipitation. Co-
transfection of these plasmids into an adenovirus packaging
cell line, preferably a cell line according to the invention,
generates recombinant replication deficient adenoviruses by a
one-step homologous recombination between the adapter and the
complementing construct.
A general protocol as outlined below, and meant as a
non-limiting example of the present invention, has been
performed to produce several recombinant adenoviruses using
various adapter plasmids and the Ad.AfIII-rITR fragment.
3o Adenovirus packaging cells (PER.C6) were seeded in "'25 cmz
flasks and the next day, when they were at "'80% confluency,
transfected with a mixture of DNA and lipofectamine agent
(Life Techn.) as described by the manufacturer. Routinely,
40,1 lipofectamine, 4~,g adapter plasmid and 4 ~g of the
complementing adenovirus genome fragment AflII- rITR (or 2 ~.g
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of all three plasmids for the double homologous
recombination) are used. Under these conditions transient
transfection efficiencies of ~50% (48 hrs post transfection)
are obtained as determined with control transfections using a
pAd/CMV-LacZ adapter. Two days later, cells are passed to ~80
cm2 flasks and further cultured. Approximately five (for the
single homologous recombination) to eleven days (for the
double homologous recombination) later a cytopathic effect
(CPE) is seen, indicating that functional adenovirus has
. io formed. Cells and medium are harvested upon full CPE and
recombinant virus is released from the cells by freeze-
thawing. An extra amplification step in a 80 cmz flask is
routinely performed to increase the yield since at the
initial stage the titers are found to be variable despite the
i5 occurrence of full CPE. After amplification, viruses are
harvested and plaque purified using PER. C6 cells. Individual
plaques are tested for viruses with active trans-genes.
Several different recombinant adenoviruses, comprising
the luciferase gene (IG.Ad.CLIP.Luc), the bacterial LacZ gene
20 (IG.Ad.CLIP.LacZ and IG.Ad.CMV.LacZ) or an empty CLIP
cassette (IG.Ad.CLIP) have been produced using this protocol.
In all cases, functional adenovirus was formed and all
isolated plaques contained viruses with the expected
expression cassettes.
B. Generation of recombinant adenoviruses deleted for
early region 1 and early region 2A
Besides replacements in the E1 region, it is possible to
delete or replace the E2A region in the adenovirus. This
3o creates the opportunity to use a larger insert or to insert
more than one gene without exceeding the maximum packagable
size (approximately 1050 of wt genome length).
Recombinant viruses that are both E1 and E2A deleted are
generated by a homologous recombination procedure as
described above for E1-replacement vectors using a plasmid-
based system consisting of:
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a) An adapter plasmid for E1 replacement according to the
invention, with or without insertion of a first gene of
interest.
b) The pWE/Ad.AfIII-rITR0E2A fragment, with or without
insertion of a second gene of interest.
Generation and propagation of such viruses, e.g.
IG.Ad.CMV.LacZ0E2A, IG.Ad.CLIP.LacZ0E2A, IG.Ad.CLIP~E2A or
IG.Ad.CLIP.Luc0E2A, requires a complementing cell line for
complementation of both E1 and E2A proteins in traps, as
to described above.
Because E3 functions are not necessary for the
replication, packaging and infection of the (recombinant)
virus, it is also possible to delete or replace (part of) the
E3 region in the E1- and/or E1/E2A-deleted adenoviral vector.
This creates the opportunity to use larger inserts or to
insert more than one gene without exceeding the maximum
packagable size (approximately 105°s of wt genome length).
This can be done, e.g., by deleting part of the E3 region in
the pBr/Ad.Bam-rITR clone by digestion with XbaI and re-
ligation. This removes Ad5 wt sequences 28592-30470 including
all known E3 coding regions. Another example is the precise
replacement of the coding region of gpl9K in the E3 region
with a polylinker allowing insertion of new sequences. This,
leaves all other coding regions intact and obviates the need
2s for a heterologous promoter since the transgene is driven by
the E3 promoter and pA sequences, leaving more space for
coding sequences.
To this end, the 2.7-kb EcoRI fragment from wt Ad5
containing the 5' part of the E3 region was cloned into the
3o EcoRI site of pBluescript (KS-) (Stratagene). Next, the
HindIII site in the polylinker was removed by digestion with
EcoRV and HincII and subsequent re-ligation. The resulting
clone pBS.Eco-Eco/ad50HIII was used to delete the gpl9K-
coding region. Primers 1 (5'-GGG TAT TAG GCC AA AGG CGC A-3')
35 and 2 (5'-GAT CCC ATG GAA GCT TGG GTG GCG ACC CCA GCG-3')
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were used to amplify a sequence from pBS.Eco-Eco/ad50HIII
corresponding to sequences 28511 to 28734 in wt Ad5 DNA.
Primers 3 (5'-GAT CCC ATG GGG ATC CTT TAC TAA GTT ACA AAG
CTA-3') and 4 (5'-GTC GCT GTA GTT GGA CTG G-3') were used on
s the same DNA to amplify Ad5 sequences from 29217 to 29476.
The two resulting PCR fragments were ligated together by
virtue of the new introduced NcoI site and subsequently
digested with XbaI and MunI. This fragment was then ligated
into the pBS.Eco-Eco/ad50HIII vector that was digested with
1o XbaI (partially) and MunI generating pBS.Eco-Eco/ad50HIII.O
gpl9K. To allow insertion of foreign genes into the HindIII
and BamHI site, an XbaI deletion was made in pBS.Eco-Eco/ad50
HIII.Ogpl9K to remove the BamHI site in the Bluescript
polylinker. The resulting plasmid pBS.Eco-Eco/ad5~HIII0gp19K
15 ~XbaI, contains unique HindIII and BamHI sites corresponding
to sequences 28733 (HindIII) and 29218 (BamHI) in Ad5. After
introduction of a foreign gene into these sites, either the
deleted XbaI fragment is re-introduced, or the insert is re-
cloned into pBS.Eco-Eco/ad50HIII.Ogpl9K using HindIII and for
2o example MunI. Using this procedure, we have generated
plasmids expressing HSV-TK, hIL-la, rat IL-3, luciferase or
LacZ. The unique SrfI and NotI sites in the pBS.Eco-Eco/ad5~
HIII. Ogpl9K plasmid (with or without inserted gene of
interest) are used to transfer the region comprising the gene
2s of interest into the corresponding region of pBr/Ad.Bam-rITR,
yielding construct pBr/Ad.Bam-rITROgpI9K (with or without
inserted gene of interest). This construct is used as
described supra to produce recombinant adenoviruses. In the
viral context, expression of inserted genes is driven by the
3o adenovirus E3 promoter.
Recombinant viruses that are both E1 and E3 deleted are
generated by a double homologous recombination procedure for
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E1-replacement vectors using a plasmid-based system
consisting of:
a) an adapter plasmid for E1 replacement according to the
invention, with or without insertion of a first gene of
interest,
b) the pWE/Ad.AflII-EcoRI fragment, and
c) the pBr/Ad.Bam-rITROgpI9K plasmid with or without
insertion of a second gene of interest.
In addition to manipulations in the E3 region, changes
to of (parts of) the E4 region can be accomplished easily in
pBr/Ad.Bam-rITR. Moreover, combinations of manipulations in
the E3 and/or E2A and/or E4 region can be made. Generation
and propagation of such vectors, however, demands packaging
cell lines that complement for E2A and/or E4 in trans.
C. Generation of E1 deleted recombinant Ad vectors that
possess an attenuated E4 region in PER.C6/tTA cells
Recombinant viruses that are E1 deleted and harbor a
synthetic E4 promoter region are generated by a homologous
2o recombination procedure as described above for E1-replacement
vectors using a plasmid-based system consisting of:
a) an adapter plasmid for E1 replacement according to the
invention, with or without insertion of a first gene of
interest,
z5 b) pWE/Ad.AfIII-rITR.tetO-E4
Generation and propagation of such viruses, e.g., IG.Ad/LacZO
EltetO-E4 is done in the appropriate complementing cells,
i.e. PERC.6/tTA cells. Several different recombinant
adenoviruses, comprising the bacterial LacZ gene
30 (IG.Ad.AdApt.LacZ and IG.Ad.ULIP.LacZ) have been produced
using this protocol (see table I).
D. Generation of recombinant Ad deleted for early region
1 and early region 2A and attenuated for E2B and/or E4
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Recombinant adenoviral vectors from which both E1 and E2A are
deleted, and which possess E2B and/or E4 regions under
transcriptional control of a synthetic promoter are generated
by homologous recombination as described above using a
combination of the following plasmid DNAs:
a) An adapter plasmid for replacement of E1, e.g., pULIP-
LacZ, pAdApt-LacZ, pEF-la-LacZ.
b) pWE/Ad.AfIII-rITR0E2Atet0-E4; pWE/Ad.AfIII-rITR0E2Atet0-
E2B; pWE/Ad.AfIII-rITR0E2Atet0-E2Btet0-E4
to Generation and propagation of such viruses is done in
the appropriate complementing cells, i.e. PER/E2A/tTA cells.
Several different recombinant adenoviruses, comprising
attenuated E2B or E4 have been produced using this protocol
(see table II).
E. Growth of Ad vectors comprising attenuated E2B or E4
in cells that do not express tTA
A selection of recombinant Ad vectors, i.e.
IG.Ad.AdApt.LacZ.DE2Atet0-E4, IG.Ad.ULIP.LacZ.DE2Atet0-E4,
2o IG.Ad.ULIP.LacZ.tetO-E2B, and IG.Ad.ULIP.LacZ.DE2A (control
virus), that were generated by the procedure described above,
were tested for their ability to replicate in PER/E2A cells
that do not express tTA. The growth of these viruses in
PER/E2A/tTA cells was analyzed in parallel. Table III shows
that the growth of IG.Ad.AdApt.LacZ.DE2Atet0-E4,
IG.Ad.ULIP.LacZ.DE2Atet0-E4, IG.Ad.ULIP.LacZ.tetO-E2B was
drastically impaired in PER/E2A cells whereas these viruses
can grow well in PER/E2A/tTA cells. This effect is not due to
differences in susceptibility of these cell lines for the
3o virus since the control virus, IG.Ad.ULIP.LacZ.DE2A, did grow
very well in PER/E2A cells. Together, this indicated that the
E2B and E4 attenuated viruses are strongly disabled in
replication in the absence of tTA despite the fact that all
other components necessary for replication were available.
Together, this result indicates that the attenuation of the
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respective gene regions (E2B and E4) according to the
invention has been successful.
Example 5
Biological activity of IG.Ad/DEltetO-E4, IG.Ad/DEl0E2Atet0-
E4, IG.Ad/DE1 4E2Atet0-E2B, and IG.Ad/DE10E2Atet0-E2Btet0-E4
vectors in vitro and in vivo.
A. Biological activity of IG.Ad/dEltetO-E4, IG.Ad/dEld
1o E2A tet0-E4, IG.Ad/dE1 dE2Atet0-E2B, and IG.Ad/dEldE2Atet0-
E2Btet0-E4 vectors in vitro
In order to demonstrate that E1 or E1+E2A deleted
recombinant Ad vectors with conditionally disabled E2B and/or
E4 genes express reduced levels of E2B and/or E4 genes, in
mammalian and/or human cells, the following experiment is
performed: HeLa cells (ATCC CCL-2) or A549 cells are seeded
at 1x106 cells per tissue culture plate (Greiner) in DMEM
(Gibco BRL) supplemented with 10°s FBS (Gibco BRL) in a l00
COz atmosphere at 37°C. The next day, cells are inoculated
2o with 0, 10, 100, 1000 or 10,000 virus particles of IG.Ad/LacZ
DEltetO-E4, IG.Ad/LacZ0E10E2Atet0-E4, IG.Ad/LacZ0E10E2Atet0-
E2B or IG.Ad/LacZ0E10E2Atet0-E2Btet0-E4 per cell. As a
control, parallel cell-cultures are inoculated with 10, 100,
1000, or 10,000 virus particles of IG.Ad/LacZ0E1 or
2s IG.Ad/LacZ0E10E2A. Forty-eight hours post inoculation, cells
can be either assayed for viral gene (E2, E4 and late genes)
expression or for LacZ expression.
The LacZ transducing efficiency is determined as
follows: Infected cells are washed twice with PBS (NPBI) and
3o fixed for 8 minutes in 0.25°s glutaraldehyde (Sigma) in PBS.
Subsequently, the cells are washed twice with PBS and stained
for 8 hours with X-gal solution (1 mg/ml X-gal in DMSO
(Gibco) , 2mM MgClz (Merck) , 5mM KQ [Fe (CN) 6] . 3H20 (Merck) , 5mM
K3[Fe(CN)6] (Merck) in PBS. The reaction is stopped by
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removal of the X-gal solution and washing of the cells with
PBS.
Expression of viral genes is assayed by 4destern blot
analysis using E2B, E4 and viral late protein specific
s antibodies using the ECL (Amersham) detection system as
described by the manufacturer.
1o B. Longevity of transgene-expression from E1 or E1+E2A
deleted recombinant Ad vectors possessing attenuated E2B
and/or E4.
In order to study whether the replacement of the native
E2B and/or E4 promoter with the synthetic promoter increases
is the longevity of expression of transgene from the recombinant
Ad vectors in vivo, the following experiments are performed.
A total of 108 or 109 virus particles of either IG.Ad/LacZO
El, IG.Ad/LacZ0E10E2A, IG.Ad/LacZOEltetO-E4, IG.Ad/LacZOEl~
E2Atet0-E4, IG.Ad/LacZ0E10E2Atet0-E2B, IG.Ad/LacZ0E10
2o E2Atet0-E2Btet0-E4 is injected into the tail vein of 8 weeks
old C57/B16 or NOD-SCID mice. At day 7, 14, 28, and 56, two
mice per group are sacrificed, the livers of these mice are
isolated and fixed in formalin. Thin slices are cut and
extensively washed in PBS. Subsequently, the slices are
2s stained in X-gal solution (1 mg/ml X-gal in DMSO (Gibco), 2mM
MgCl2 (Merck) , 5mM K4 [Fe (CN) 6] . 3H20 (Merck) , 5mM K3 [Fe (CN) 6]
(Merck) in PBS. After an 8-hour incubation, the samples are
washed in PBS. The longevity of LacZ expression from the
different Ad vectors will thus be assayed. It is expected
3o that the expression of LacZ in C57/B16 mice is prolonged when
replication-conditioned Ad vectors were used instead of the
conventional IG/Ad.LacZ4E1 and IG/Ad.LacZ0E10E2A vectors. In
contrast, the LacZ expression in the livers of immune-
deficient NOD-SCID mice is expected to be stable in all
35 cases .
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Example 6
Residual E4 gene expression from IG.Ad/~E1, IG.Ad/DE10E2A,
IG.Ad/DEltetO-E4, and IG.Ad/DE10E2Atet0-E4 vectors in vitro
5
In order to demonstrate that El- or E1+E2A-deleted
recombinant Ad vectors with conditionally disabled E4 genes
express reduced levels of E4 genes in mammalian and/or human
cells, the following experiment was performed: A549 cells
to were seeded at 1x106 cells per 10 cm2 tissue culture dish
(Greiner) in DMEM (Gibco BRL) supplemented with 10% FBS
(Gibco BRL), and incubated in a loo C02 atmosphere at 37°C.
The next day, the cells were inoculated with 1000 virus
particles of IG.Ad/AdAptLuc0El, IG.Ad/AdAptLuc0E10E2~1,
15 IG.Ad/AdAptLucOEltetO-E4, or IG.Ad/AdAptLuc0E10E2Atet0-E4.
The cells were harvested at 30 h post-inoculation by lysis in
100 ~,1 RIPA buffer (PBS + to NP40 + 0.5o deoxycholic acid +
O.lo SDS + protease inhibitor cocktail). After 15 min
incubation on ice, the cell lysates were spun for 15 min at
20 14,000 rpm, 4°C in an eppendorf centrifuge. The total protein
concentration in the supernatants was thereafter determined
using the Bio-Rad DC Protein Assay. The (relative) amounts of
the E4-orf6 (34kD) protein present in the cell extracts were
determined by western blot analysis. For this purpose, equal
25 amounts (30 ~,g) of total protein from the cleared cell
extracts were run in an SDS-polyacrylamide gel and thereafter
blotted onto an Immobilon-P membrane (Millipore). This
membrane was processed by incubation with a rabbit E4-orf6
specific anti-peptide antiserum (first antibody, 1:2000
3o diluted; Boivin et al., 1999) and a blotting grade affinity
purified Goat anti-Rabbit IgG (H+L)-HRP (secondary antibody,
1:7500 diluted; Biorad). The E4-orf6 protein was eventually
visualized using the ECL PlusTM Western blotting detection
reagents (Amersham Pharmacia Biotech) according to the
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_ 61
manufacturer's recommendations. The data in Fig.8 and 9
clearly show that the E1- and E1+E2A-deleted Ad vectors with
conditionally disabled E4 genes produced significantly less
of the E4-orf6 protein than E1- or E1+E2A-deleted vectors
s possessing wt E4. This indicates that the expression of E4,
in non-complementing cells, i.e., normal mammalian and/or
human cells, is significantly reduced by the replacement of
the native E4 promoter by the tet operon.
io To demonstrate that the reduced expression of E4 was not
simply due to a difference in transduction efficiency of the
various vectors, a semi-quantitative southern blot analysis
of the cell-associated viral genomes was performed.
Therefore, total DNA was harvested from the infected cells at
15 30 h post-inoculation by using the Easy-DNA kit (Invitrogen)
according to the manufacturer's recommendations. Ten ~g of
each DNA sample was digested with BamHI, and run in a 0.750
agarose gel. The DNA was thereafter blotted onto a HybondTM
N+ nylon transfer membrane (Amersham Pharmacia Biotech) and
2o probed with a HindIII-NheI (484 bp) fragment of the Ad5 fiber
gene that was labeled with 32P-CTP using the Rad Prime RTS
System (GIBCO). Although some variation in the amount of
vector DNA could be observed (Fig.lO), it is clear that the
reduced expression of E4 from IG.Ad/AdApt.Luc0E1tet0-E4, and
25 IG.Ad/AdAptLuc~E10E2Atet0-E4 cannot be explained by
inefficient transduction. For example, despite the relatively
low abundance of viral genomes of the El+E2A deleted vector,
this vector produced significantly more E4-orf6 protein than
the vectors possessing conditionally disabled E4. Taken
so together, these results provide evidence that the attenuation
of E4 according to the invention leads to a significant
reduction of E4 expression in normal mammalian and/or human
cells.
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Example 7
Residual E2 gene expression from IG.Ad/~E1, IG.Ad/~E10E2A,
IG.Ad/~E1~E2Atet0-E4, and IG.Ad/DE10E2Atet0-E4 vectors in
vitro
In order to demonstrate that E1- or El+E2A-deleted
recombinant Ad vectors with conditionally disabled E4 genes
express reduced levels of the E2A (DBP) gene in mammalian
io and/or human cells, the following experiment was performed:
A549 cells were seeded at a density of 1x106 cells per 10 cm2
tissue culture dish (Greiner) in DMEM (Gibco BRL)
supplemented with 10o FBS (Gibco BRL), and incubated in a l00
C02 atmosphere at 37°C. The next day, the cells were
inoculated with 1000 virus particles of IG.Ad/AdAptLucOEl,
-~IG.Ad/AdAptLuc0E10E2A, IG.Ad/AdAptLuc0El0E2AtetO-E4, or
IG.Ad/AdAptLuc0E10E2AtetO-E4. At 30 h post-inoculation, the
cells were harvested as described supra and the cell extracts
were analyzed for the presence of the E2A protein DBP by the
2o western blot assay as described supra, except that the
membrane was processed by incubation with the anti-DBP
monoclonal antibody B6 (first antibody, 1:1000 diluted; Reich
et al., 1983) and a blotting grade affinity purified Goat
anti-Mouse IgG (H+L)-HRP (secondary antibody, 1:7500 diluted;
z5 Biorad) .
The results (Fig.l1) clearly show that the E1-deleted Ad
vector with conditionally disabled E4 produced significantly
reduced amounts of the DBP protein in comparison to the E1-
3o deleted vector possessing wt E4. As expected, the El+E2A-
deleted vectors produced no DBP protein. Taken together,
these results provide evidence that the attenuation of E4
according to the invention leads to a significant reduction
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of E2A expression in normal mammalian and/or human cells.
B) In order to demonstrate that E1- or E1+E2A-deleted
recombinant Ad vectors with conditionally disabled E4 genes
express reduced levels of E2B genes in mammalian and/or human
cells, the following experiment was performed: A549 cells
were seeded at a density of 1x106 cells per 10 cm2 tissue
culture dish in DMEM supplemented with 10% FBS, and incubated
in a loo C02 atmosphere at 37°C. The next day, the cells were
to inoculated with 1000 and 10000 virus particles of IG.Ad/DE1,
IG.Ad/DE10E2A, or IG.Ad/DE10E2Atet0-E4. The cells were
harvested at 30h post-inoculation and the relative amount of
the E2B (p)TP protein present in the cell extracts was
determined by Western blot analysis as described above except
that this time, a mixture (1:1:1) of three antibodies against
(p)TP and Pol (kind gift of P. C. van der Vliet, Utrecht, the
Netherlands) was used as primary antiserum (1:500 diluted).
The data shown in Fig.l2 clearly demonstrate that the E1- and
E1+E2A-deleted Ad vectors with conditionally disabled E4
2o genes produced significantly reduced amounts of the pTP
protein than E1 or E1+E2A deleted vectors possessing wt E4.
Example 8
Residual late gene expression from IG.Ad/DEl, IG.Ad/~E10E2A,
IG.Ad/DE10E2Atet0-E4, and IG.Ad/DEl0E2Atet0-E4 vectors in
vitro.
In order to demonstrate that E1-deleted Ad vectors with
conditionally disabled E4 genes express reduced levels of
late genes in mammalian and/or human cells, the following
experiment was performed: A549 cells were seeded at 1x106
cells per 10 cm2 tissue culture dish in DMEM supplemented
with 10o FBS, and incubated in a loo C02 atmosphere at 37°C.
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The next day, the cells were inoculated with 1000 virus
particles of IG.Ad/AdAptLuc0El, IG.Ad/AdAptLuc0E10E2A,
IG.Ad/AdAptLuc0El0E2Atet0-E4, or IG.Ad/AdAptLuc0E10E2Atet0-
E4.
s The cells were harvested at 72 h post-inoculation and the
relative amount of the fiber protein present in the cell
extracts was determined by western blot analysis as described
above except that the polyclonal E641/3 anti-knob domain of
fiber (primary antibody, 1:5000 diluted; kind gift of R.
1o Gerard, Leuven, Belgium) and the blotting grade affinity
purified Goat anti-Rabbit IgG \(H+L)-HRP (secondary antibody,
1:100000 diluted; Biorad) were used. The results in Fig. l3
clearly show that the El- and E1+E2A-deleted Ad vectors with
conditionally disabled E4 genes, as well as the vector that
15 was deleted of E1+E2A produced significantly reduced amounts
of fiber protein than the vector that was deleted of E1 only.
This shows that attenuation of the expression of the E4 genes
by itself causes a reduction in late gene expression, a
phenomenon that is also seen in Ad vectors that are deleted
20 of E2A.
Example 9
Attenuation of E4 expression leads to a diminished liver
toxicity in vivo.
To demonstrate that Ad vectors with conditionally disabled E4
are less toxic in vivo than Ad vectors possessing wt E4, mice
are injected intravenously via the tail vein with 1E11 vp of
IG.Ad/DE1, IG.Ad/~El0E2A, IG.Ad/~EltetO-E4, IG.Ad/DE10
3o E2Atet0-E4, or with PBS/0.5o sucrose only. For this purpose,
as a non-limiting example, BALB/c, C57BL/6 and C3H mice are
used. Ten mice per vector and per time-point are used. All
vectors are suspended in PBS/0.5% sucrose, and except for
modifications in the E2A and E4 regions, these vectors are
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genetically identical and lack a transgene and accompanying
transcription elements to avoid any unintentional effect of a
transgene and/or accompanying transcription elements on liver
toxicity. The mice are sacrificed on day 3, 14, 28, 56 and 90
s after injection, weighed and the livers of these mice are
isolated and weighed as well. Removal of liver: liver and
gall bladder are removed. Of each liver, a part of the
anterior and posterior right lobe is cut-off with a clean
scalpel and snap frozen in liquid nitrogen and processed for
to real-time PCR or Southern blot analysis (to check the
transduction efficiency). The remaining liver lobes (median,
left and caudate) are fixed in ample buffered formalin. The
median lobe (with biliary cyst) and left lobe are trimmed at
their largest cross section for HDS staining.~The livers are
15 examined histologically. Liver lesions, such as vacuolar
change, apoptosis, dense nuclei, inclusions, mitotic
increase, anisonucleosis, megalocytosis, and inflammation in
peri-portal and sinusoidal areas are scored semi-
quantitatively. In addition, blood is sampled for blood cell
2o counts (erys, leucocytes, thrombocytes) and plasma is taken
for biochemical measurement of ALAT, ASAT, AP, gamma-GT, ALB
and TBIL. All procedures are executed according to procedures
very well known to persons skilled in the art.
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Example 10
The effect of the attenuation of E4 on the activity of the
CMV promoter driving a transgene is cell-type specific
In order to determine the effect of the attenuation of E4 on
the activity of the CMV promoter the following experiments
have been performed. A549 cells were seeded at 1x106 cells
per 10 cm2 tissue culture dish in DMEM supplemented with 10%
FBS, and incubated in a 10% COZ atmosphere at 37°C. The next
io day, the cells were inoculated with 1000 virus particles of
IG.Ad/AdAptLuc0El, IG.Ad/AdAptLuc0E10E2A, IG.Ad/AdAptLucO
EltetO-E4, or IG.Ad/AdAptLuc0E10E2AtetO-E4. Notably, all
these vectors contain the luciferase gene under the control
of the CMV promoter. The cells were harvested by detergent-
mediated lysis at 48 h post-inoculation and the luciferase
activity in the cell extracts was measured and expressed in
RLU (relative light units) using the Luciferase Assay System
(Promega) according to the manufacturer's recommendation. The
RLU was normalized to the total amount of protein in the cell
2o extracts, which was measured by using the BioRad DC Protein
Assay. The results, as shown in Fig.l4, indicate that the Ad
vectors possessing conditionally disabled E4 produced
significantly less luciferase than the vectors possessing wt
E4. This implies that the CMV promoter that drives the
expression of the luciferase gene was less active in vectors
possessing conditionally disabled E4 than in vectors
possessing wt E4.
To find out whether the inhibitory effect of the attenuation
of E4 on the CMV promoter activity also occurs in other cell
3o types, an experiment, similar to the one described above, was
done using primary human endothelial cells. These cells were,
for this purpose, inoculated with 1000 vp of IG.Ad/AdAptLucO
E1 or 1000 vp of IG.Ad/AdAptLuc0E10E2AtetO-E4. Forty-eight
hours after inoculation, cell extracts were made and examined
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as described above. The results (Fig. l5) show that similar
amounts of luciferase were found in cells infected with the
Ad containing wt E4 and the one possessing conditionally
disabled E4. This indicates that the activity of the CMV
promoter is not impaired by the attenuation of E4 in primary
human epithelial cells. From these experiments, it is
concluded that the effect of the attenuation of E4 on the
activity of the CMV promoter is cell type specific.
to Example 11
The influence of E4 in transgene expression over longer
periods of time in lung-, liver and breast tumor derived cell
lines
To determine whether E4-expression plays a role in transgene
expression from the CMV-promoter over longer periods of time
in lung and liver derived cell-lines, the following
infection- and subsequent luciferase activity experiments
2o were performed. As a part of these experiments, total protein
content was also determined.
Transduction of A549 (lung derived) cells
At day l, A549 cells were seeded in 96-well plates with a
density of 10,000 cells/well in a volume of 100 ~1 DMEM+l0o
heat-inactivated FBS and incubated in a humidified COZ
incubator set at 37°C and 10o CO2. At day 2, the cells were
transduced with viruses derived from PER/E2A/tTA cells
transfected with the adapter plasmid pAdApt-luc in
3o combination with pWE/Ad.AfIII-rITR0E2Atet0-E4 or derived from
PER.C6/E2A cells that were transfected with pAdApt-Luc in
combination with pWE/Ad.AflII-rITR0E2A. In this transduction
experiment crude lysates as well as purified viruses were
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used. For both types of preparations the number of virus
particles (vp) per ml were determined. Infections were
performed for 5 time points in quadruplate per time point.
The Multiplicities of Infections (MO T s) were 5,000 and
s 50,000 vp/cell in a total volume of 150 u1. After
transduction, the cells were again incubated in a humidified
COz incubator set at 37°C and 10o C02, At day 3, cells in
plate for time point "24 hour" were washed with 100 u1 PBS
and lysed with 100 u1 lysisbuffer (8 mM MgCl2, 1 mM EDTA, 1
to mM DTT, to v/v Triton X-100 and 15% v/v glycerol). Then the
plate was frozen at -20°C until luciferase- and total protein
assays could be performed.
At day 4, cells in plate for time point "48 hour" were
washed, lysed and stored according to the protocol described
i5 supra. Medium of plates for time points "72 hour", "96 hour"
and "168 hour" was removed and new medium was added, to avoid
re-infection. At day 5, 6 and 9, cells in plates for
respectively time points "72 hour", "96 hour" and "168 hour"
were washed, lysed and stored according the protocol
2o described supra.
Luciferase experiments were performed as follows. After
thawing, the plates were centrifuged for 5 min at 1500 rpm
and put on ice. Luciferase expression was determined with a
luminometer [EG&G Berthold]. For this, 20 u1 sample was put
2s in an appropriate tube after which the machine added 100 u1
luciferase assay substrate buffer (luciferase assay substrate
dissolved in 10m1 luciferase assay buffer [Promega Catno.
E1501]). Some samples were diluted in lysisbuffer because
expression was too high to measure.
3o To correct for total protein quantity in the samples, the
CBQCA protein quantitation assay (Molecular Probes. Catno. C-
6667) was performed according the manufacturers protocol. For
all samples, 5 u1 was used in the assay.
Fig. l6 shows the results of the transduction of the A549
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cells. These data show that the DE2A.tetO-E4 viruses (normal
lines) give about a 100 fold lower expression over time as
compared to the DE2A viruses (dashed lines). There is no
clear decrease in luciferase expression for each virus
s separately over time. After 168 hours the level of luciferase
activity using these different viruses (purified or crude) is
comparable to levels detected after 24 and 48 hours.
Transduction of HepG2 (liver derived) cells
to Transduction, luciferase and protein content determination
experiments for HepG2 cells were performed according to the
protocol described supra for A549 cells, with the following
two exemptions. HepG2 cells were seeded in 96-well plates
with a density of 22,500 cells/well (100 u1) and the MOT s
i5 that were used were 30 and 300 vp/cell in a total volume of
150 u1.
Fig. l7 shows the luciferase activity results obtained after
the transduction of the HepG2 cells. The results suggest that
expression over time of the ~E2A.tetO-E4 (normal lines) and
2o the ~E2A viruses (dashed lines) are comparable. The
luciferase activity derived from both viruses apparently
increase over time.
Transduction of MCF-7 (breast cancer derived) cells
25 Transduction, luciferase and protein content determination
experiments for MCF-7 cells were performed according to the
protocol described supra for A549 cells, with the exemption
that the medium used was DMEM containing 10% non-heat
inactivated FBS.
3o Fig. l8 shows the luciferase activity results obtained after
the transduction of the MCF-7 cells. These results suggest
that expression over time of the ~E2A.tetO-E4 (normal lines)
and the DE2A viruses (dashed lines) are also comparable. The
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result obtained after the DE2A virus infection using a crude
lysate with an MOI of 50,000 vp/cell, suggest that there is a
drop in expression level after 72 hours. The results obtained
after infection with DE2A.tetO-E4 using crude lysates and
s purified viruses show a slight decrease in luciferase
activity after 96 hours.
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Example 12
Genomic analysis of Ad vectors with conditionally disabled E4
The genomic identity of Ad vectors possessing conditionally
s disabled E4 was confirmed by Southern blot analysis of the
vector genome. For this purpose, the viral genomic DNA was
isolated from purified IG.Ad/DE1~E2A and IG.Ad/~E10E2AtetO-
E4 virus particles. Therefore, 100 ~1 virus suspension
containing 1.4x1011 - 3x1011 virus particles was mixed with 18
to ~l buffer (50 mM MgCl2, 1.2 mM CaCl2, and 130 mM Tris pH
7.5), 9 ~,l DNaseI (10 mg/ml), and 3 ~1 H20. This mixture was
incubated for 30 min at 37°C after which 3.6 ~1 EDTA (0.5 M),
4.5 ~1 SDS (10%), and 1.5 ~l Proteinase K (20 mg/ml) was
added. The mixture was then incubated for 1 h at 50°C. The
15 viral DNA was then purified from the mixture using the
GeneClean Kit and cut with PstI. Equal amounts of DNA
purified from different vectors were run in a to agarose gel
and blotted onto a HybondTM-N+ nylon transfer membrane and
probed with a 313 by HindIII/NcoI fragment of pNEB-PaSe.tet0.
2o This fragment corresponds to the tet operon sequence and was
labeled with 32P-CTP using the Rad Prime RTS System (GIBCO).
The data in Fig. l9 show that only a fragment of the genomic
DNA of IG.Ad/DE1~E2AtetO-E4 was labeled whereas the genomic
DNA of IG.Ad/~E10E2A was unlabeled. This indicates that only
25 the genomic DNA of IG.Ad/~E10E2AtetO-E4 contained the tet
operon sequence. The size of the labeled fragment fits the
theoretic length of the genomic PstI fragment (2203 bp)
containing the rITR and sequences of E4 with the tet operon.
From this result, it is concluded that IG.Ad/DEl0E2AtetO-E4
3o truly possesses a tet operon in place of the native E4
promoter.
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Example 13
Serum-free suspension cultures of PER/E2A/tTA and PER.C6/tTA
cells.
s To obtain a serum-free suspension culture of PER/E2A/tTA
cells (clone 1A1, pn 11), 5x106 cells were thawed and
cultured in ExCell 525 medium (JRH Bioscienses) supplemented
with 4mM L-glutamin with or without the addition of
Hygromycin (100 ug/ml). Cells were cultured in T175 flasks.
io The concentration of Hygromycin is identical to what is used
for adherent cultures. However, since the the ExCell 525
medium does not support the culturing of PER.C6 cells and
derivatives thereof in the presence of Hygromycin, culturing
was continued only with cultures in the absence of the
15 Hygromycin selection pressure. Cultures that were kept in the
presence of Hygromycin died after 8 days.
Culture conditions in the absence of Hygromycin were as
follows: Passage of the cells was done after 2 or 3 days of
incubation. A sample of the cells was subsequently taken and
2o counted and stained for determination of the cell density and
viability of the cultures. Then, cultures were passed to a
new flask or to a roller bottle or diluted in the flask in
which the culture was kept. Following this, the culture was
sub-cultured to 2x105 or to 3x105 viable cells per ml and
2s left for another incubation period of 3 or 2 days, depending
on the concentration of cells.
The cell cultures'were further incubated at 37°C or at
39°C.
The standard temperature for incubating adherent cell
cultures that contain a temperature sensitive E2A gene (like
3o PER/E2A/tTA) is 39°C. The static cultures (in flasks) were
incubated at 39°C, while the dynamic cultures in roller
bottles were incubated at 37°C.
After 13 population doublings a serum free suspension cell
bank of 5x106 cells/vial was established. After 11 and 15
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population doublings, infections using recombinant LacZ
expressing and E4-attenuated adenovirus in roller bottles
were performed. Roller bottles contained 100 ml suspension
cultures with a cell density of approximately 1x106 viable
s cells per ml. These infections yielded functional LacZ
expressing virus as was determined after re-infection of the
supernatants on A549 cell cultures and subsequent staining of
the cells.
The dynamic serum free suspension culture was maintained in
io roller bottles for 61 days, with a total of 44 population
doublings, which results in an average population doubling
time of 33 hours. The static serum free suspension culture in
the flasks was maintained for 34 days, with a total of 19
population doublings, which results in an average population
15 doubling time of 42 hours.
Two 2-liter bio-reactor runs were also performed. In the
first bio-reactor a run was performed with cells that were
kept in the reactor for 8 days with a final density of
approximately 4x106 cells per ml using standard perfusion
2o conditions known to persons skilled in the art.
In the second bio-reactor, cells were cultured for 4 days
after which a virus infection was performed while the cell
density was approximately 1x106 cells per ml. For this an MOI
of 70 virus particles per cell was applied with purified
2s attenuated-E4 recombinant adenovirus. After 4 days of
infection the bio-reactor run was terminated and the cells
and medium were harvested.
The experiments to obtain a serum free suspension culture of
3o PER.C6/tTA were similarly executed. For this 5x106 PER.C6/tTA
cells (clone 2C5 pn 8) were cultured in ExCell 525 medium
supplemented with 4 mM L-glutamin. These cells
adapted to serum free suspension medium after 2 passages
after thawing.
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7.4
Culture conditions were as follows: Passaging of the cells
was done after 2 or 3 days of incubation. A sample of the
cell culture was used for counting and staining for
determination of the cell density and viability of these
s cultures. The culture was then passed to a roller bottle or
diluted in the same flask as it was cultured in. The culture
was diluted to 2x105 or 3x105 viable cells per ml for an
incubation period of 3 or 2 days respectively. All roller
bottle (dynamic) cultures were incubated at 37°C and flushed
io with C02.
After 18 population doublings an infection in roller bottles
containing 5x105 viable cells per ml was performed. The
infection was performed with an attenuated-E4 recombinant
adenovirus. The obtained virus titer from this infection
15 (taken three days after infection) was 1.2x101° virus
particles per ml. This number equals 24,000 produced virus
particles per cell (seeded cells).
After 22 population do_ublings a serum free suspension cell
bank of 5x106 cells per vial was established. This was
2o performed after.27 days of culturing resulting in an average
population doubling time of 29 hours.
Two 2-liter bio-reactor runs were also performed with the
PER.C6/tTA cells. In the first bio-reactor cells were
cultured for 8 days and reached a density of approximately
2s 4x106 cells per ml using standard perfusion known to persons
skilled in the art. In the second bio-reactor cells reached a
density of approximately 3x106 cells per ml and were
subsequently infected with a concentrated batch of
recombinant attenuated-E4 adenovirus that was derived from
3o the infected roller bottle cultures described supra. An MOI
of 70 virus particles per cell was applied, using standard
perfusion methods. The culture was kept at 37°C. Three days
after infection the virus yield in crude samples taken from
the bio-reactor was measured. The concentration of this virus
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stock was 7x101° virus particles per ml. This equals a
production rate of approximately 23,000 virus particles per
cell.
5
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Example 14
Plaque purification, propagation, and titration of Ad vectors
comprising conditionally disabled E4
A.
Plaque purification: IG.Ad/DEltetO-E4 and IG.Ad/~E10E2Atet0-
E4 vectors were plaque-purified by using PER.C6/tTA and
PER/E2A/tTA cells, respectively. For this purpose, cells were
seeded in 6-wells plates at a density of 1.5x106 cells per 10
io cm2 well in DMEM + 10% FBS + 10 mM MgCl2. After a 4 h
incubation at 37°C, 10% C02 (PER.C6/tTA) or 39°C, 10% C02
(PER/E2A/tTA) the medium was replaced by 1 ml of inoculation
medium. The inoculation medium consisted of IG.Ad/DEltetO-E4
or IG.Ad/DE1~E2Atet0-E4 vectors that had been diluted in DMEM
+ 10% FBS + 10 mM MgCl2. Usually, 10-fold dilutions were made
ranging from 10-9 to 10-9. The cells were incubated in
inoculation medium o/n at 37°C, loo COZ (PER.C6/tTA) or 34°C,
COZ (PER/E2A/tTA). Thereafter, the inoculation medium was
removed and the cells were washed with PBS and overlaid with
3 ml MEM + 10 mM MgCl2 + 2.5o agarose + 2-5% FBS per well.
The cells were further incubated at 37°C, 10% C02
(PER.C6/tTA) or 34°C, 10% C02 (PER/E2A/tTA). Eleven to 14
days later, independent, free plaques were picked using a 20-
~l pipette. Twenty ~1 of the plaque material was resuspended
z5 in 200 ~1 DMEM + 10°s FBS + 10 mM MgCl2, and frozen at -20°C.
B.
Propagation of plaque-purified vectors: the above-described
resuspended plaque material containing IG.Ad/DEltetO-E4 or
3o IG.Ad/DE10E2Atet0-E4 was thawed and 100 ~,1 of each plaque
material was mixed with 900 ~1 of DMEM + loo FCS + 10 mM
MgCl2, and used to inoculate sub-confluent monolayers of
PER.C6/tTA or PER/E2A/tTA cells in 2.5 cm2 wells,
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respectively. Cells were incubated at 37°C, loo C02
(PER.C6/tTA) or at 34°C, loo C02 (PER/E2A/tTA). Full CPE
usually occurred after 4 to 7 days. Thereafter, the cells
were scraped from the dish and harvested together with the
s medium. The cell/medium suspension was then frozen at -20°C.
After thawing, 0.5 ml of this suspension was used to
inoculate sub-confluent monolayers of.fresh cells in 80 cmZ-
culture flasks (Nunc) for further amplification. This
procedure could be repeated and scaled up to large-scale
Zo vector propagations. High yields (more than 25,000 vp per
cell) of progeny vectors were obtained when fresh cells were
inoculated at an MOI of 50-200 vp per cell.
C.
1s Determination of vector titers: PER.C6/tTA and PER/E2A/tTA
cells were used to determine the infectious titer of batches
of IG.Ad/DEltetO-E4 and IG.Ad/DE10E2Atet0-E4, respectively.
For this purpose, cells were seeded in 96-wells at. a density
of 4x10 cells per well in DMEM + loo FBS + 10 mM MgCl2. The
2o medium was replaced after a 4-hr incubation at 37°C, loo C02
(PER.C6/tTA) or 39°, loo COZ (PER/E2A/tTA) by 200 ~1 of
serial dilutions (made in DMEM + 10% FBS + 10 mM MgCl2) of
IG.Ad/~EltetO-E4 or IG.Ad/DE10E2Atet0-E4, respectively.
Cells were further incubated at 37°C, loo COZ and 34°C, l00
2s C02, respectively, and CPE was monitored every 3-4 days.
After 14-16 days, the titer of the vectors was calculated
from the highest dilution of vector that gave CPE.
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Example 15
Stability of PER/E2A/tTA cells in producing E1+E2A-deleted Ad
vectors that are conditionally disabled in E4
s PER/E2A/tTA (clone 1A1) cells were kept in culture for at
least 100 passages in medium containing the selection drug
hygromycin (100 ~g/ml). Routinely, the cells were split (1:3
- 1:5) twice per week. To verify that PER/E2A/tTA cells
maintain their capacity to efficiently support the
io propagation of El+E2A-deleted, E4-attenuated Ad vectors the
propagation of such vectors was compared in PER/E2A/tTA cells
that had been kept in culture for a long and short period.
For this purpose, 2.5 x 106 cells PER/E2A/tTA cells at
passage 17 and at passage 64 were seeded in a 25 cm2 culture
is flask (Nunc) in DMEM + 10 o FBS + 10 mM MgCl2, and incubated
at 39°C, 10o COz. The next day, the cells were inoculated
with IG.Ad/AdAptLacZ0E10E2Atet0-E4 at an MOI of 200 vp/cell.
After a 5-days incubation at 34°C and loo COz, the progeny
vectors were harvested by freeze-thawing the cells and
2o medium. Thereafter, 10-fold dilutions, ranging from 10-1 to
10-6, of the progeny vectors were made in DMEM + loo FBS, and
used to inoculate sub-confluent monolayers of A549 cells in
2.5 cm2 wells. After a 48 h incubation at 37°C, the cells
were washed twice with PBS and fixed for 8 min with 1%
2s formaldehyde, 0.2o glutar(di)aldehyde in PBS. The cells were
thereafter stained with X-gal solution (1 mg/ml X-gal in DMSO
(Gibco) , 2 mM MgCl2 (Merck) , 5 mM KQ [Fe (CN) 6] . 3H20 (Merck) , 5
mM K3[Fe(CN)6] (Merck) in PBS). The reaction was stopped by
removal of the X-gal solution and washing the cells with PBS.
3o The percentage of blue cells was thereafter determined. The
results (see Table V) revealed that the progeny vectors
derived from PER/E2A/tTA at passage 17 and 64 yielded very
similar percentages of blue cells at the various dilutions.
This indicates that the yields of progeny vector from
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PER/E2A/tTA cells at passage 18 and 65 were very similar.
This implies that PER/E2A/tTA cells maintain their capacity
to support the propagation of E1+E2A-deleted, E4-attenuated
vectors over an extensive period of time during which they
s are kept in culture.
Example 16
Construction of pAd/pIPspAdapt-eGFP, pAd/pIPspAdapt-lacZ,
pAd/pIPspAdapt-ceNOS, pAd/pIPspAdapt-hIL3, pAd/pIPspAdapt-
Zo hEPO and pAd/pIPspAdapt-LacZ
pAdS/L420-HSA (described in published PCT patent application
WO 99/55132) was digested with AvrII and BglII. The vector
fragment was ligated to a linker oligonucleotide digested
is with the same restriction enzymes. The linker was made by
annealing oligos of the following sequence: PLL-1 (5'- GCC
~ATC CCT AGG AAG CTT GGT ACC GGT GAA TTC GCT AGC GTT AAC GGA
TCC TCT AGA CGA GAT CTG G-3') and PLL-2 (5'- CCA GAT CTC GTC
TAG AGG ATC CGT TAA CGC TAG CGA ATT CAC CGG TAC CAA GCT TCC
2o TAG GGA TGG C-3'). This ligation resulted in pAdMire.
Another batch of pAdS/L420-HSA was also digested with AvrII
and 5' protruding ends were filled in using Klenow enzyme. A
second digestion with HindIII resulted in removal of the L420
promoter sequences. The vector fragment was isolated and
2s ligated separately to a PCR fragment containing the CMV
promoter sequence. This PCR fragment was obtained after
amplification of CMV sequences from pCMVLacI (Stratagene)
with the following primers: CMVplus (5'-GAT CGG TAC CAC TGC
AGT GGT CAA TAT TGG CCA TTA GCC-3') and CMVminA (5'-GAT CAA
3o GCT TCC AAT GCA CCG TTC CCG GC-3'). The PCR fragment was
first digested with PstI after which the 3'-protruding ends
were removed by treatment with T4 DNA polymerase. Then the
DNA was digested with HindIII and ligated into the
AvrII/HindIII digested pAdS/L420-HSA vector. The resulting
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plasmid was named pAdS/CMV-HSA. This plasmid was then
digested with HindIII and BamHI and the vector fragment was
isolated and ligated to the HindIII/BglII polylinker sequence
obtained after digestion of pAdMire. The resulting plasmid
s was named pAdApt.
The full length human EPO cDNA (Genbank accession number:
M11319) was cloned, employing oligonucleotide primers EPO-
START:S' AAA AAG GAT CCG CCA CCA TGG GGG TGC ACG AAT GTC CTG
CCT G-3' and EPO-STOP:5'AAA AAG GAT CCT CAT CTG TCC CCT GTC
1o CTG CAG GCC TC-3' (Cambridge Bioscience Ltd) in a PCR on a
human adult liver cDNA library. The amplified fragment was
cloned into pUCl8 linearized with BamHI. Sequence was checked
by double stranded sequencing. The full length human EPO cDNA
containing a perfect Kozak sequence for proper translation
15 was removed from the pUCl8 backbone after a BamHI digestion.
The cDNA insert was purified over agarose gel and ligated
into pAdApt which was also digested with BamHI, subsequently
dephosphorylated at the 5' and 3' insertion sites using SAP
and also purified over agarose gel to remove the short BamHI-
zo BamHI linker sequence. The obtained circular plasmid was
checked with KpnI, DdeI and NcoI restriction digestions that
all gave the right size bands. Furthermore, the orientation
and sequence were confirmed by double stranded sequencing.
The obtained plasmid with the human EPO cDNA in the correct
2s orientation was named pAdApt.EPO and was further digested
with HindIII and XbaI restriction enzymes. This EPO insert
was isolated and ligated to a HindIII/XbaI digested
pIPspAdapt6 plasmid (described in WO 99/64582). The resulting
plasmid was named pAd/pIPspAdapt-hEPO. pIPspAdapt6 plasmids
3o carrying human ceNOS (insert HindIII/XbaI), human IL-3
(insert HindIII/BamHI), LacZ (insert KpnI/BamHI) and eGFP
(insert HindIII/EcoRI) were generated via pAdS/CLIP
(described in WO 99/55132) and according to methods described
in detail in WO 99/64582.
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Example 17 -
Miniaturized, multiwell production of El/E2A deleted and E4
attenuated recombinant adenoviral vectors in PER/E2A/tTA
s cells
Several different PER/E2A/tTA cell clones were tested for the
production of recombinant adenoviral viruses with E1 and E2A
deletions in combination with an E4 attenuation. This was
to performed in 96 well tissue culture plates, to test for
usefulness in high-through-put screens in functional
genomics.
First set of transfections
15 At day 1, eighteen different PER/E2A/tTA cell clones were
harvested and diluted in culture medium (DMEM+10% Fetal
Bovine Serum and 10 mM MgClz) to a density of 22,500 cells
per 100 ~,1. These suspensions were seeded in two 96-well=
tissue-culture plates with 100 ~,1 per well in duplo (one
2o clone in four wells divided over two plates). At day 2, one
DNA mix was made for each PER/E2A/tTA clone, by diluting 3.9
~,g of SalI linearized pAd/Adapt-LacZ and 3.9 ~g of PacI
linearized pWE/Ad.AfIII-rITR0E2A.tetO-E4, in 150 ~1 DMEM. To
each DNA mix, 150 ~1 Lipofectamine mix (38.4 ~1 Lipofectamine
25 (Life Techn.) + 111.6 ~1 DMEM) was added. This
DNA/Lipofectamine mixture was left at room temperature for 30
min, followed by the addition of 1.95 ml DMEM. The latter
mixtures were then added (30 ~l per well) to the PER/E2A/tTA
cells, after removal of the medium in which the cells were
3o seeded. After 2 hours incubation in a humidified C02
incubator (39°C, loo COZ), 170 ~l culture medium was added to
each well and the plates were returned to the humidified C02
incubator (39°C, 10% COz). At day 3, the supernatant in each
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well was replaced with 200 ~1 culture medium. The plates were
then placed again in another humidified COZ incubator (34°C,
loo COZ). At day 5, one of the duplo 96-wells plates was used
to determine the transfection efficiency using LacZ staining
s (Table III). The LacZ staining procedures are described in
PCT/W099/64582. The other plate was monitored for CPE
formation during a period of three weeks after transfection.
In Fig.20 the percentage of CPE positive wells, scored three
weeks after transfection, is depicted. These results suggest
1o that it was best to continue with PER/E2A/tTA clones 1A1,
1C1, 1C3, 2B3, 2C3 and 2D5 for 96 well adenoviral production
settings, as outlined supra. These clones were used in a
second round transfection procedure.
15 Second set of transfections
At day 1, six attached PER/E2A/tTA cell cultures (1A1, 1C1,
1C3, 2B3, 2C3 and 2D5) were harvested and diluted in culture
medium (DMEM+10% FBS and 10 mM MgCl2) to a density of 22,500
cells per 100 ~1. Subsequently, two 96-well-tissue culture
2o plates for each clone were used to. seed 100 ~1 of the cell
suspensions per well. At day 2, three different DNA mixes
were prepared for each PER/E2A/tTA clone. 1 ~,g of a
linearized adapter molecule (being either pAd/pIPspAdapt-
eGFP, or pAd/pIPspAdapt-lacZ or pAd/Adapt-ceNOS) was mixed
25 with 4 ~g of PacI linearized pWE/Ad.AfIII-rITR~E2A.tetO-E4 in
100 ~,1 DMEM. To each mix 100 ~,1 Lipofectamine mix (25.6 ~1
Lipofectamine (Life Techn.) + 74.4 ~,1 DMEM) was added. This
DNA/Lipofectamine mixture was left at room temperature for 30
min, after which 1.3 ml DMEM was added. The latter mixtures
3o were then added on top to the PER/E2A/tTA cells in a total
volume of 30 ~1 per well, after removal of the medium in
which the cells were seeded. After 2 hours incubation in a
humidified COZ incubator (39°C, loo C02), 170 ~l culture
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medium was added to each well and the plates were placed back
in the humidified C02 incubator (39°C, 10% COz). At day 3, the
medium of each well was replaced with 200 ~1 culture medium.
The plates were then returned to another humidified C02
s incubator (34°C, loo COZ). At day 5, one of the two 96-wells
plates of each clone that was transfected with the LacZ
adapter plasmid was used to determine the transfection
efficiency using LacZ staining as described supra. Table IV
shows the transfection efficiency of each clone. The other
io plate for the LacZ transfectants and all other plates were
monitored for CPE formation during a period of three weeks
after transfection. In Fig.21 the percentage of CPE positive
wells, scored three weeks after transfection, is depicted.
i5 Example 18
Generation of pBr/Ad.Bam-rITR0E2Atet0-E4.DE3(XbaI) and
pWE/Ad.AfIII-rITR0E2Atet0-E4.DE3(XbaI)
To construct E3 deleted versions of the vectors carrying a
2o tet0-E4 attenuation, the following cloning steps were
performed. pBr/Ad.Bam-rITR0E2Atet0-E4 was propagated in E.
coli strain DMl (dam-, dcm-) (Life Techn.). The purified-
plasmid was digested with Xbal, hereby removing the 1.88 kb
Xbal-Xbal insert, and subsequently relegated. By removing the
2s Xbal-Xbal insert the following sequences were deleted: 191 by
of the E3-6.7K protein, the E3-19K glycoprotein, the E3-ADP
(10.5K protein), RIDalpha (E3-10.4K protein), RIDbeta (E3-
14.6K protein) and the first 21 by of the E3-14.7K protein
(for sequences, see Genbank accession number X03002). The
3o resulting plasmid was named pBr/Ad.Bam-rITR0E2Atet0-E4.0
E3(XbaI) and was used to construct helper cosmid
pWE/Ad.AflII-rITR~E2Atet0-E4.DE3(XbaI). pBr/Ad.Bam-rITRO
E2Atet0-E4.DE3(XbaI) was digested with BamHI and PacI
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restriction enzymes and an 11 kb fragment, with the Xba1-Xbal
deletion, was isolated. The plasmid pWE/Ad.AfIII-rITR0E2A was
first digested with BamHI and then partially digested with
PacI, yielding amongst others a 26.2 kb fragment. The 11 kb
s BamHI-PacI fragment from and the 26.2 kb fragment were
ligated, yielding cosmid pWE/Ad.AfIII-rITR~E2Atet0-E4.~
E3(XbaI). This cosmid contains sequences identical to that of
pWE/Ad.AfIII-rITR0E2Atet0-E4 but with the deletion of the
XbaI-XbaI fragment. pWE/Ad.AfIII-rITR0E2Atet0-E4.DE3(XbaI)
to was used in the production of adenoviruses with E1, E2A and
E3 deletions and E4 attenuations in 96 well plates.
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Example 19
Comparison between pWE/Ad.AfIII-rITR0E2Atet0-E4 and
pWE/Ad.AflII-rITR~E2Atet0-E4.DE3(XbaI) in the generation of
E4 attenuated viruses in a 96-wells setting for the purpose
5 of functional genomics.
At day 1, PER/E2A/tTA clone 1C3 was harvested and diluted
with culture medium (DMEM+10% FBS and 10 mM MgCl2) to a
density of 22,500 cells per 100 ~1, followed by seeding 100
l0 1 per well in 96-well-tissue culture plates. At day 2,
linearized adapter molecules pAd/pIPspAdapt-ceNOS,
pAd/pIPspAdapt-eGFP, pAd/pIPspAdapt-hEPO, pAd/pIPspAdapt-
hIL3, pAd/pIPspAdapt-lacZ and pAd/pIPspAdapt-luciferase were
used for transfection in combination with 2 different PacI
15 linearized helper cosmids: pWE/Ad.AflII-rITR0E2Atet0-E4 and
pWE/Ad.AfIII-rITR0E2Atet0-E4.DE3(XbaI).
The DNA transfection procedure was identical to that
described in Example (above) describing transfections in a
subset of PER/E2A/tTA clones. The transfection efficiency of
2o wells, transfected with lacZ as adapter molecule, scored
after lacZ staining, was 70-80o for both cosmid combinations.
CPE formation was monitored during a period of three weeks
after transfection. Fig.22 shows the percentage of CPE
positive wells in a comparison between pWE/Ad.AfIII-rITR~
2s E2Atet0-E4 and pWE/Ad.AflII-rITR0E2Atet0-E4.DE3(XbaI)
transfections. Subsequently, the wells were subjected to
freezing in the culture medium at -20°C, followed by thawing
and resuspension by repeated pipetting. An aliquot of 100 ~,1
of the freeze/thawed transfected cells was transferred to
3o each well of a plate with freshly seeded and attached
PER/E2A/tTA cells of clone 1C3. These cells were seeded as
described above. The second 96-well plate with PER/E2A/tTA
cells, incubated with the freeze/thawed cell lysates of the
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first transfected plate, was checked for CPE formation and
stored at -20°C. Fig.23 shows the percentage of virus
propagation (CPE positive wells) in 96-wells plates seeded
with fresh PER/E2A/tTA cells. This was scored after infection
s with the supernatants from the freeze/thawed transfected
cells as shown in Fig.22. The generation of functional virus
using this set-up was shown by several assays (described in
WO 99/64582) in which the presence of the proteins that are
encoded by the adapter plasmids was determined. Fig.24 shows
1o the percentage of functional viruses that produce either
human IL-3, LacZ or Luciferase in the 96-wells setting.
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Example 20
Determination of toxicity using tet0-E4 attenuated viruses in
comparison to non-tet0-E4 attenuated viruses using microarray
technology
To determine the different effects on cellular gene
expression and vector associated toxicity between adenoviral
vectors with different deletions and attenuations, microarray
experiments are performed. This allows the measurement of
io mRNA expression of thousands of genes simultaneously and the
effect a particular adenoviral vectors has on these gene
expressions.
On day 1, different cell lines, primary as well as
i5 established, are seeded in T25 tissue culture flasks. The
next day cells are infected with E1, E1/E2A, E1/E2A/E3
deleted or E4 attenuated adenoviral vectors with a serie of
different MOT s. After 24 hours, medium of the infected cells
is changed. Then, 24, 48 and 72 hours after infection cells
2o are harvested and trizol lysates are made from which RNA and
subsequently cDNA is generated. This cDNA is used to generate
Cy3 and Cy5 labeled probes using common techniques, known to
those skilled in the art and described in Zhu et al (1998).
Using the wild type E1 deleted Ad5 vector as a reference,
z5 expression profiles are determined for adenoviral vectors
with different deletions and attenuations by hybridizing the
labeled probes to microarrays (reviewed in Marshall and
Hodgson 1998; Ramsay 1998). The fluorescent signals of both
Cy3 and Cy5 are determined using a microarray scanner and
3o converted using controls for hybridization and cDNA labeling
and generation to normalized fluorescent values. The Cy3 and
Cy5 values are then compared which results in relative data
showing the difference in response of cells to infection with
different types of adenoviral vectors including tet0-E4
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attenuated and non tet0-E4 attenuated vectors.
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Example 21
In vivo production of nitric oxid upon treatment with E4
attenuated viruses expressing ceNOS.
s The in vivo duration of NOS3 transgene expression after
aerosol gene transfer of rat lungs with the recombinant
adenovirus IG.Ad/DE10E2Atet0-E4-ceNOS was assessed.
Twelve male Wistar rats (body weight 300350 g) were
aerosolized via a silastic catheter into the trachea with
1o IG.Ad/DE10E2Atet0-E4-ceNOS recombinant adenovirus (300 ~l
physiologic salt solution containing 3x109 plaque forming
units over 60 minutes). After viral delivery, the catheter
was removed from the trachea and animals were extubated. No
side effects were observed during aerosol delivery or
i5 following extubation.
At several time points (day 3, 7, 14 and 21) the transduced
animals were re-anaesthetized and re-intubated for exhaled
Nitric Oxide measurements during room air breathing. Nitric
Oxide (NO) levels in exhaled air were measured using standard
2o chemiluminescence (Sievers 280 - NO analyzer NOATM). Each day
of exhaled NO measurements, calibration was performed using a
calibration-gas-mixture of exactly 400 parts per million
(ppm). The minimal detectable NO concentration was 1 ppb.
Two animals died 24 hours after the aerosolisation (170),
2s probably due to traumatic intubation and persistent tracheal
damage after extubation. Mean body weight of the transfected
animals at baseline was 363~27 g and decreased to 319~15 g
(P<0.05 vs baseline) three days after aerosolisation. Seven
days after singe aerosol gene transfer, body weight was
3o normalized (325~15 gr, P=NS vs baseline). Three days after
aerosolisation, the exhaled NO levels of the aerosolized
animals increased from baseline (5~1 ppb) to 14~5 ppb (n=3).
Exhaled NO levels remained elevated at day 7 and 14: 12~9
(n=2) and 16~5 ppb (n=4) respectively. After 21 days exhaled
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NO levels returned back to baseline levels, which is
approximately 5~1 ppb.
These results suggest that there is a longer duration of
expression of ceNOS using IG.Ad/DE1~E2Atet0-E4-ceNOS virus as
s compared to the duration of ceNOS expression using a first
generation (non-attenuated E4) adenoviral ceNOS virus, which
was used in the acute and prolonged hypoxia model (Budts et
al. 2000). In this model, first generation adenoviral vectors
express ceNOS only for one week and the measured
to concentration of exhaled N0, as determined at day 7 and day
14 returns to baseline levels at day 14. No toxicity of
clinical relevance and differences in loss of body weight as
compared to non-E4 attenuated vectors, that were used in the
past, were detected after gene transfer with the attenuated-
i5 E4 adenovirus.
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FIGURE AND TABLE LEGENDS
Figure 1: (A) Expression of DBP, Penton and Fiber.
A549 cells were infected with a multiplicity of infection
(m.o.i.) of either 0, 100, 1,000 or 10,000 vp/cell of
IG.Ad/CLIP or IG.Ad.CLIP0E2A. Seventy-two hours post
infection, cell extracts were prepared and equal amounts of
whole cell extract were fractionated by SDS-PAGE in 10% gels.
The proteins were visualized with the aDBP monoclonal B6,
io the polyclonal a-Penton base Ad2-Pb571 or the polyclonal a-
knob domain of fiber E641/3, using an ECL detection system.
Cells infected with IG.Ad.CLIP express both E2A encoded DBP,
Penton base and Fiber proteins. The proteins co-migrate with
the respective proteins in the positive control (lane P,
i5 extract from PER. C6 cells infected with IG.Ad.CLIP harvested
at starting CPE). In contrast, no DBP, penton-base or fiber
was detected in the non-infected A549 cells or cells infected
with IG.Ad.CLIP0E2A. These data show that deletion of the E2A
gene did not only eliminate residual DBP expression, but also
2o the residual expression of the late adenoviral proteins ,
penton-base and fiber.
(B) Residual expression of E4-orf6 and pTP/TP.
The blot, as shown in A, was stripped and used to visualize
the E4-orf6 and pTP/TP proteins using the method described
25 above but now by using a monoclonal antibody against E4-orf6
or a polyclonal anti-pTP/TP antiserum, respectively. These
proteins co-migrate with the respective proteins in the
positive control (P). The results show that pTP/TP as well as
E4-orf6 are still produced from the E1/E2A deleted Ad vector.
so This indicates that the deletion of the E2A gene did not
eliminate the residual expression of the E2B and E4 genes.
Figure 2: Temperature dependent growth of PER. C6.
PER. C6 cells were cultured in Dulbecco's Modified Eagle
35 Medium supplemented with 10% Fetal Bovine Serum (FBS, Gibco
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BRL) and lOmM MgClz in a 10~ C02 atmosphere at either 32°C,
37°C or 39°C. At day 0, a total of 1 x 106 PER. C6 cells were
seeded per 25cmz tissue culture flask (Nunc) and the cells
were cultured at either 32°C, 37°C or 39°C. At day 1-8,
cells
were counted. The growth rate and the final cell density of
the PER.C6 culture at 39°C are comparable to that at 37°C.
The growth rate and final density of the PER.C6 culture at
32°C were slightly reduced as compared to that at 37°C or
39°C.
to PER.C6 cells were seeded at a density of 1 x 106 cells per 25
cm2 tissue culture flask and cultured at either 32-, 37- or
390C. At the indicated time points, cells were counted in a
Burker cell counter. PER.C6 grows well at both 32-, 37- and
390C.
Figure 3: DBP levels in PER. C6 cells transfected with pcDNA3,
pcDNA3wtE2A or pcDNA3ts125E2A.
Equal amounts of whole-cell extract were fractionated by
SDS-PAGE on 10°s gels. Proteins were transferred onto
2o Immobilon-P membranes and DBP protein was visualized using
the aDBP monoclonal B6 in an ECL detection system. All of
the cell lines derived from the pcDNA3ts125E2A transfection
express the 72-kDa E2A-encoded DBP protein (left panel,
lanes 4-14; middle panel, lanes 1-13; right panel, lanes 1-
12). In contrast, the only cell line derived from the
pcDNAwtE2A transfection did not express the DBP protein (left
panel, lane 2). No DBP protein was detected in extract from a
cell line derived from the pcDNA3 transfection (left panel,
lane 1), which serves as a negative control. Extract from
3o PER. C6 cells transiently transfected with pcDNA3ts125 (left
panel, lane 3) served as a positive control for the Western
blot procedure. These data confirm that constitutive
expression of wtE2A is toxic for cells and that using the
ts125 mutant of E2A can circumvent this toxicity.
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Figure 4: Suspension growth of PER.C6ts125E2A C5-9.
The tsE2A expressing cell line PER.C6tsE2A.c5-9 was cultured
in suspension in serum free Ex-cellT"". At the indicated time
points, cells were counted in a Burker cell counter. The
results of 8 independent cultures are indicated. PER.C6tsE2A
grows well in suspension in serum free Ex-cellT"" medium.
Figure 5: Growth curve PER. C6 and PER.C6tsE2A.
1o PER.C6 cells or PER.C6ts125E2A (c8-4) cells were cultured at
37°C or 39°C, respectively. At day 0, a total of 1 x 106 cells
was seeded per 25cm2 tissue culture flask. At the indicated
time points, cells were counted. The growth of PER. C6 cells
at 37°C is comparable to the growth of PER.C6ts125E2A c8-4 at
39°C. This shows that constitutive overexpression of ts125E2A
has no adverse effect on the growth of cells at the non-
permissive temperature of 39°C.
Figure 6: Stability of PER.C6ts125E2A.
2o For several passages, the PER.C6ts125E2A cell line clone
8-4 was cultured at 39°C in medium without 6418. Equal
amounts of whole-cell extract from different passage numbers
were fractionated by SDS-PAGE on 10°s gels. Proteins were
transferred onto Immobilon-P membranes and DBP protein was
2s visualized using the aDBP monoclonal B6 in an ECL detection
system. The expression of ts125E2A encoded DBP is stable for
at least 16 passages, which is equivalent to approximately 40
cell doublings. No decrease in DBP levels were observed
during this culture period, indicating that the expression of
3o ts125E2A is stable, even in the absence of 6418 selection
pressure.
Figure 7: tTA activity in hygromycin resistent PER.C6/tTA (A)
and PER/E2A/tTA (B) cells.
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Sixteen independent hygromycin resistent PER.C6/tTA cell
colonies and 23 independent hygromycin resistent PER/E2A/tTA
cell colonies were grown in 10 cmz wells to sub-confluency
and transfected with 2 ~g of pUHC 13-3 (a plasmid that
contains the reporter gene luciferase under the control of
the 7xtet0 promoter). One half of the cultures was maintained
in medium containing doxycycline to inhibit the activity of
tTA. Cells were harvested at 48 hours after transfection and
luciferase activity was measured. The luciferase activity is
to indicated in relative light units (RLU) per ~tg protein.
Fig.8
Western blot to visualize the residual expression of E4-orf6
(E4-34 kDa) protein and a Southern blot to visualize the
i5 cell-associated viral DNA. A549 cells were inoculated with
1000 vp/cell of IG.Ad/AdAptLuc~El (dEl), IG.Ad/AdAptLuc0E10
E2A (dE2A), IG.Ad/AdAptLuc0EltetO-E4 (dEl.tet0-E4), or
IG.Ad/AdAptLuc0E10E2AtetO-E4 (dEl.dE2A.tetO-E4). At 30 h
post-inoculation, the cells were harvested and the relative
2o amount of E4-orf6 protein in each sample was determined by
Western blotting using an E4-orf6 specific anti-peptide serum
(kind gift of Dr P. Branton). Parallel cultures, infected by
the same vectors at the same time, were used to check the
transduction efficiency of the vectors. This was done by
25 Southern analysis.
Fig.9
The infections (described for Fig.8) for each analysis were
done in triplicate and analyzed by western blotting.
Fig.lO
The infections (described for Fig.8) for each analysis were
done in triplicate and analyzed by Southern blotting.
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Fig. l1
A549 cells were inoculated with 1000 vp/cell of
IG.Ad/AdAptLuc~El (dE1), IG.Ad/AdAptLuc0E10E2A (dE2A),
IG.Ad/AdAptLuc0E1tet0-E4 (dEl.tet0-E4), or IG.Ad/AdAptLuc~El
s DE2Atet0-E4 (dEl.dE2A.tetO-E4). All infections were performed
in triplicate. At 30 h post-inoculation, the cells were
harvested and the relative amount of DBP protein in each
sample was determined by Western blotting using the anti-DBP
monoclonal antibody B6 (first antibody, 1:1000 diluted Reich
to et al., 1983). In parallel, one culture was analyzed that not
been infected with any vector (mock). As a positive control,
a lysate of cells in which an Ad vector had undergone
replication, had been taken.
15 Fig. 12
A549 cells were inoculated with 1000 vp per cell using E1-
deleted Ad vectors (dEl), E1+E2A-deleted Ad vectors (dE2A),
E1-deleted and E4-attenuated (dEl.tet0-E4), or E1+E2A-deleted
and E4-attenuated (dEl.dE2A.tetO-E4). At 30 h post-
2o inoculation, the cells were harvested and the relative amount
of the E2B encoded protein pTP in each sample was determined
by Western blotting using a mixture (1:1:1) of three
antibodies against (p)TP and Pol.
25 Fig. l3
A549 cells were inoculated with 1000 vp/cell of
IG.Ad/AdAptLuc0E1 (dEl), IG.Ad/AdAptLuc~E10E2A (dE2A),
IG.Ad/AdAptLuc0E1tet0-E4 (dEl.tet0-E4), or IG.Ad/AdAptLuc~El
~E2Atet0-E4 (dEl.dE2A.tetO-E4). All infections were performed
3o in triplicate. At 72 h post-inoculation, the cells were
harvested and the relative amount of fiber protein in each
sample was determined by Western blotting using the
polyclonal E641/3 anti-knob domain of fiber (primary
antibody, 1:5000 diluted).
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96
Fig. l4
A549 cells were inoculated with IG.Ad/AdAptLuc0E1 (dEl),
IG.Ad/AdAptLuc~E10E2A (dEl.dE2A), IG.Ad/AdAptLuc~EltetO-E4
(dEl.tet0-E4), or IG.Ad/AdAptLuc~E10E2Atet0-E4
(dEl.dE2A.tetO-E4). All infections were performed in
triplicate and using the indicated multiplicity of infection
(MOI). The cells were harvested by detergent-mediated lysis
at 48h post-inoculation and the luciferase activity in the
io cell extracts was measured and expressed in relative light
units (RLU) per ~,g protein present in the cell extracts.
Fig. l5
Primary human endothelial cells were inoculated with
s5 IG.Ad/AdAptLuc~El0E2A (dE2A) or with IG.Ad/AdAptLuc0E10
E2Atet0-E4 (dE2A.tetO-E4). All infections were performed in
triplicate and using the indicated multiplicity of infection
(MOI). The cells were harvested by detergent-mediated lysis
at 48h post-inoculation and the luciferase activity in the
2o cell extracts was measured and expressed in relative light
units (RLU) per ~g protein present in the cell extracts.
Fig. l6
Longevity of expression in A549 cells of Luciferase upon
2s infection with crude lysates and purified virus in a
comparison study with attenuated-E4 and non-attenuated-E4
recombinant adenoviruses.
Fig. l7
Longevity of expression in HepG2 cells of Luciferase upon
infection with crude lysates and purified virus in a
comparison study with attenuated-E4 and non-attenuated-E4
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97
recombinant adenoviruses.
Fig. l8
Longevity of expression in MCF-7 cells of Luciferase upon
s infection with crude lysates and purified virus in a
comparison study with attenuated-E4 and non-attenuated-E4
recombinant adenoviruses.
Fig. l9
io Viral genomic DNA was isolated from equal numbers of purified
E1+E2A-deleted (Ad.dE2A) and E1+E2A-deleted, E4-attenuated
(Ad.dE2A.tetO-E4) Ad vector particles and cut with PstI. As
positive controls, the plasmid pWE/Ad.AfIII-rITR.tetO-E4 was
digested with PacI and PstI, and the plasmid pNEB-PaSe.tet0
15 was digested with HindIII. The DNA samples were run in a is
agarose gel, blotted onto a membrane and probed with a 32P
~labeled 313 by HindIII/NcoI fragment of pNEB-PaSe.tet0.
Fig.20
2o Percentage of CPE positive wells, based on the results of the
first transfection round. CPE was scored three weeks after
transfection.
Fig.21
2s Percentage of CPE positive wells in the second transfection
round, scored three weeks after transfection in six different
PER/E2A/tTA clones transfected with either pAd/pIPspAdapt-
eGFP, or pAd/pIPspAdapt-lacZ or pAd/Adapt-ceNOS. All three
different transfections were in addition to pWE/Ad.AfIII-rITR
3o DE2A.tetO-E4.
Fig.22
Percentage of cpe positive wells in 96-wells plates using
PER/E2A/tTA cells transfected with lacZ adapter plasmid in
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98
combination with either pWE/Ad.AflII-rITR0E2Atet0-E4 or
pWE/Ad.AfIII-rITR0E2Atet0-E4.DE3(XbaI).
Fig.23
Percentage of cpe positive wells in 96-wells plates using
fresh PER/E2A/tTA cells infected with supernatants from
PER/E2A/tTA cells that were transfected with lacZ adapter
plasmid in combination with either pWE/Ad.AflII-rITR0E2Atet0-
E4 or pWE/Ad.AfIII-rITR0E2Atet0-E4.DE3(XbaI).
to
Fig.24
Percentage of cpe positive wells harboring viruses that
produce functional hIL-3, LacZ and Luciferase.
Table I: Generation of E2B and E4 attenuated recombinant
adenoviral vectors.
This table shows a selection of E2B and E4 attenuated Ad
vectors that have been generated by cotransfection of the
2o indicated plasmid DNAs into the indicated cells. A cytopathic
effect (CPE) typical for adenovirus replication (the
appearance of rounded cells and so-called comets in the cell
monolayer) is usually detected at 8-14 days after
transfection. Transfection of only one of the plasmids did
not result in CPE (not shown).
Table II A and H: Growth of E2B and E4 attenuated recombinant
adenoviral vectors in PER.C6/E2A and PER/E2A/tTA cells.
The indicated viruses were diluted as indicated (from 10-3 -
10-') and used to inoculate subconfluent monolayers of the
indicated cells grown in 10 cm2 wells. Parallel cultures of
the cells were not incubated with virus (mock). The cells
were incubated at 34°C. CPE was scored at 4 (A) and 5 (B)
days after inoculation. 0 means no CPE; 0-1 means sporadic
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, 99
comet formation; 1 means several comets present in the
culture, 25% of the cells show CPE; 2 means 50% of the cells
shows CPE ; 3 means 75% of the cells show CPE; 4 means 100%
of the cells show CPE.
s
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CA 02384439 2002-03-08
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CA 02384439 2002-03-08
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0 3
CA 02384439 2002-03-08
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110
Table =It
Traszsfection efficiency. in different PER/E2A/tTA clones,
using pAd/Adapt-,,LaCZ~in combination with pWE/Ad.AfIII~-
rITRAE2A.tetO-E4 determined upon LacZ staining. pn= passage
numrier .
p~R/E~A/tTA Blue
Clone coloxed cells
1A1 pn 16. 50-60
1A1 pn 63 60
1A3 20-50
1A4 40-50
1A6 20-40
1H1 5-20
1H4 5-40
1B5 10-20
1C1 50-60
1C2 . 40
1C3 30-40
1C4 10-50
1D4 10
1D6, 10-20
2A4 10-20
2A5 20-30
2B3 20-60
2c3 60
2D5 10-20
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Table IV
Traasfection efficiency of six different PER/E2A/tTA clones
transfected with pAd/pIPspAdapt-lacZ and gWE/Ad.AflII-
rITR~E2A.tetO-E4 using LaCZ staining. pn= passage number
PER/~~A/tTA Blue
C7.oae colored cells
1A1 pn 16 5
lCl 5
1C3 50
283 60
2C3 20
2D5 30
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112
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