U.S. patent application number 09/920932 was filed with the patent office on 2002-06-13 for helper viruses for the preparation of recombinant viral vectors.
Invention is credited to Lusky, Monika, Mehtali, Majid.
Application Number | 20020072120 09/920932 |
Document ID | / |
Family ID | 9481555 |
Filed Date | 2002-06-13 |
United States Patent
Application |
20020072120 |
Kind Code |
A1 |
Lusky, Monika ; et
al. |
June 13, 2002 |
Helper viruses for the preparation of recombinant viral vectors
Abstract
Novel helper vectors are provided for complementing defective
recombinant viral vectors, characterized in that they are provided
with recombination sequences recognized by a recombinase. A
complementation cell expressing the recombinase, and a method for
preparing recombinant viral vectors as infectious viral particles
for transferring and expressing genes of interest in a host
organism or cell, are also provided. The invention is particularly
suitable for use in gene therapy, especially in humans.
Inventors: |
Lusky, Monika; (Freiburg,
DE) ; Mehtali, Majid; (Illkirch Graffenstaden,
DE) |
Correspondence
Address: |
Norman H. Stepno
BURNS, DOANE, SWECKER & MATHIS, L.L.P.
P.O. Box 1404
Alexandria
VA
22313-1404
US
|
Family ID: |
9481555 |
Appl. No.: |
09/920932 |
Filed: |
August 3, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09920932 |
Aug 3, 2001 |
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09563239 |
May 2, 2000 |
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09563239 |
May 2, 2000 |
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09011257 |
Mar 9, 1998 |
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09011257 |
Mar 9, 1998 |
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PCT/FR96/01200 |
Jul 30, 1996 |
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Current U.S.
Class: |
435/457 ;
435/235.1; 435/325; 435/69.1 |
Current CPC
Class: |
C12N 7/00 20130101; C12N
2710/10343 20130101; C12N 15/86 20130101; C12N 2710/10352 20130101;
C12N 2800/30 20130101 |
Class at
Publication: |
435/457 ;
435/235.1; 435/69.1; 435/325 |
International
Class: |
C12N 015/861; C12N
007/00; C12P 021/02; C12N 005/06 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 31, 1995 |
FR |
95 09289 |
Claims
1. A helper virus for the production of a recombinant viral vector
defective for replication, characterized in that it comprises a
first recombination sequence at 5' and a second recombination
sequence at 3' of a region essential to the propagation of said
helper virus; said recombination sequences being recognized by a
recombinase.
2. A helper virus according to claim 1, characterized in that it is
deficient for replication.
3. A helper virus according to claim 1 or 2, characterized in that
said recombination sequences, are positioned at 5' and 3' of the
encapsidation region of said virus.
4. A helper virus according to one of claims 1 to 3, characterized
in that said first and second recombination sequences are
positioned in the same orientation with respect to one another.
5. A helper virus according to one of claims 1 to 4, characterized
in that said first and second recombination sequences are selected
from the group formed by the sequences loxP, FRT and R.
6. A helper virus according to one of claims 1 to 5, for the
production of a recombinant adenoviral vector defective for
replication, which derives from the genome of an adenovirus and
comprises the ITRs 5' and 3', an encapsidation region and at least
one viral gene selected from the genes of the E1, E2, E4 and L1-L5
regions, said gene being defective in said recombinant adenoviral
vector, characterized in that it comprises a first recombination
sequence at 5' and a second recombination sequence at 3' of said
encapsidation region.
7. A helper virus according to claim 6, characterized in that it is
devoid of all or part of the E1 and/or E4 region.
8. A helper virus according to one of claims 1 to 7, characterized
in that it additionally comprises a DNA fragment coding for a
recombinase.
9. A complementation cell line comprising a DNA fragment coding for
a recombinase.
10. A complementation cell line according to claim 9 and a helper
virus according to claim 8, in which said DNA fragment codes for a
recombinase selected from the group formed by CRE, CRE-ER, FLP and
R.
11. A complementation cell line according to claim 9 or 10,
deriving from the 293 line.
12. A procedure for preparing a viral particle comprising a
recombinant viral vector, which comprises the following steps: (a)
Preparing a recombinant viral vector-deficient for replication; (b)
Preparing a helper virus according to one of claims 1 to 8 and 10;
(c) Introducing the recombinant viral vector and the helper virus
into an appropriate cell line; (d) Culturing said cell line under
appropriate conditions to allow the production of the viral
particle in the presence of a functional recombinase able to
recognize said first and second recombination sequences; and (e)
Recovering the viral particle in the cell culture.
13. A procedure according to claim 12, which additionally comprises
an amplification step, probably in step (d).
14. A procedure according to claim 12 or 13, according to which, in
the course of step (d), the recombinase is added to the cell
culture.
15. A procedure according to claim 12 or 13, according to which
said helper virus is according to claim 8 and said cell line is the
293 complementation cell line.
16. A procedure according to claim 15, which comprises the
following steps: (a) Preparing a recombinant adenoviral vector
defective for all of the adenoviral functions with the exception of
E4; (b) Preparing a helper virus defective for the E1 and E4
functions and comprising (i) the LoxP sequences at 5' and at 3' of
the encapsidation region and (ii) a DNA fragment coding for the
CRE-ER hybrid recombinase; (c) Introducing the recombinant viral
vector and the helper virus into a 293 cell line; (d) Culturing
said cell line under appropriate conditions to allow the production
of the viral particle and, after a sufficiently long time,
continuing the culture in a medium comprising estradiol; and (e)
Recovering the viral particle in the cell culture.
17. A procedure according to claim 12 or 13, according to which
said helper virus is according to one of claims 1 to 7 and said
cell line according to one of claims 9 to 11.
18. A procedure according to claim 17, which comprises the
following steps: (a) Preparing a recombinant adenoviral vector
defective for all of the adenoviral functions with the exception of
E4; (b) Preparing a helper virus defective for the E1 and E4
functions and comprising the LoxP sequences at 5' and 3' of the
encapsidation region; (c) Introducing the recombinant viral vector
and the helper virus into a 293 cell line comprising a DNA fragment
coding for the CRE-ER hybrid recombinase. (d) Culturing said cell
line under appropriate conditions to allow the production of the
viral particle and, after a sufficiently long time, continuing the
culture in a medium comprising estradiol; and (e) Recovering the
viral particle in the cell culture.
19. A procedure according to one of claims 12 to 18, which
additionally comprises a step of purification of the recombinant
viral vector.
20. A procedure for preparing a viral particle comprising a
recombinant viral vector by means of a helper virus, according to
which the ratio viral particles of recombinant viral vector to
helper virus is greater than 50%, advantageously greater than 60%,
preferably greater than 70% and, in a very preferred manner greater
than 80%.
21. A viral particle obtained by a procedure according to one of
claims 12 to 20.
22. A eukaryotic host cell comprising a viral particle according to
claim 21.
23. A pharmaceutical composition comprising as therapeutic or
prophylactic agent a viral particle according to claim 21 or a
eukaryotic host cell according to claim 22, in combination with a
carrier acceptable from a pharmaceutical point of view.
24. Viral particle according to claim 21 or eukaryotic host cell
according to claim 22 for use as medicament.
Description
[0001] The present invention relates to novel helper vectors
allowing defective recombinant viral vectors, which have the
characteristic of being provided with recombination sequences
recognized by a recombinase, to be complemented. It likewise
relates to a complementation cell expressing the recombinase as
well as a method of preparation of recombinant viral vectors in the
form of infectious viral particles allowing the transfer and the
expression of genes of interest in a cell or a host organism. The
invention is of very particular interest for gene therapy
prospects, especially in man.
[0002] The possibility of treating human diseases by gene therapy
has passed in the course of a few years from the stage of
theoretical considerations to that of clinical applications. The
first protocol applied to man was initiated in the United States in
September 1990 on a patient who was genetically immunodeficient
because of a mutation affecting the gene coding for adenine
deaminase (ADA). The relative success of this first experiment
encouraged the development of new gene therapy protocols for
various genetic or acquired diseases (infectious diseases and
especially viral diseases such as AIDS or cancers). The great
majority of the protocols described until now employ viral vectors
to transfer and express the therapeutic gene in the cells to be
treated.
[0003] The interest in adenoviruses as gene therapy vectors has
already been touched on in numerous documents of the prior art. In
fact, the adenoviruses have a wide spectrum of hosts, are not very
pathogenic and do not have the disadvantages connected with the
retroviruses since they are nonintegrative and replicate equally in
quiescent cells. By way of information, their genome is formed of a
linear and double-stranded DNA molecule of approximately 36 kb
carrying regions acting in cis (ITR 5' and 5' encapsidation region
of the viral genome and ITR 3') and additionally about thirty
genes, at the same time early genes necessary for viral replication
and late structure genes (see FIG. 1).
[0004] The early genes are divided into 4 regions dispersed in the
adenoviral genome (E1 to E4; E for early). They comprise 6
transcriptional units which have their own promoters. The late
genes (L1 to L5; L for late) partly cover the early transcription
units and are, for the majority, transcribed starting from the
major late promoter MLP.
[0005] At the present time, all the adenoviral vectors used in gene
therapy protocols are devoid of the major part of the E1 region
essential for replication, in order to avoid their distribution in
the environment and the host organism (first generation vectors).
This deletion makes the viral genome deficient for replication.
However, the E1 viruses can be propagated in a cell line which
complements the E1 function to generate an infectious viral
particle. The 293 line, established starting from human embryonic
kidney cells, is currently used, in the genome of which is
integrated the left 5' end of the type 5 adenovirus (Graham et al.,
1977, J. Gen. Virol. 36, 59-72).
[0006] The majority of adenoviral vectors of the prior art comprise
supplementary deletions. Certain of these have been introduced in
the E3 region with the aim of increasing the cloning capacities but
do not need to be complemented to the extent where the E3 region is
nonessential for replication. More recently, second generation
vectors have been proposed in the literature. They conserve the in
cis regions (ITRs and encapsidation sequences) and comprise
important internal deletions aimed at suppressing the main part of
the viral genes whose expression can be responsible for
inflammatory responses in the host. In this respect, a minimal
vector which is deficient for all of the coding viral regions
represents a choice alternative.
[0007] The techniques of preparation of adenoviral vectors are
widely described in the literature. Firstly, the complete genome is
formed by homologous recombination in the 293 line (see especially
Graham et Prevect, 1991, Methods in Molecular Biology, Vol. 7, Gene
Transfer and Expression Protocols; Ed E. J. Murray, The Human Press
Inc. Clinton, N.J.) or in Escherichia coli (technique described in
the French Application No. 94 14470).
[0008] It is then necessary to propagate the vector in order to
form a stock of viral particles containing it. This production step
is critical and must allow high titers of infectious particles to
be attained to be able to consider development on a large scale
with a view to the preparation of clinical batches. If the first
generation adenoviral vectors can be propagated relatively easily
in the 293 cell line, the only complementation line described to
date and capable of efficiently expressing E1, such is not the case
for second generation vectors. In fact, according to the same basic
principle, such a vector must be complemented for the essential
functions which it cannot express.
[0009] The complementation can be provided "in trans" by or the
cell line employed (designated complementation cell line). It is
then necessary to have new lines complementing several essential
viral functions (E1 and E2, E1 and E4 or E1, E2 and E4). However,
the various attempts carried out until now give the impression that
the co-expression of several adenoviral regions is potentially
toxic, such that the line risks not being optimal in terms of
growth capacity and yield of viral particles, these two criteria
being indispensable for industrial exploitation.
[0010] Another alternative is based on the use of a supplementary
viral element, called "helper virus" introduced into the line at
the same time as the adenoviral vector (two-component system). At
the present time, an adenovirus from which the E1 region has been
deleted and which is capable of synthesizing the expression
products of other adenoviral regions is currently used. The
co-transfection of such a helper virus and of an adenoviral vector
in the 293 line allows the formation of viral particles.
[0011] However, a major disadvantage of this method is that the
cells produce a mixed population of viral particles, one type
comprising the recombinant vector and the other type the helper
virus. In practice, the preparations mainly contain viral particles
of helper virus, the contamination being able to reach and even
exceed 90%. The presence of the helper virus is not desirable in
the context of a therapy applied to man and therefore necessitates
the employment of cumbersome and costly physical separation
techniques, such as ultracentrifugation. In addition, this
technology is not very well adapted to the production of vectors of
complex structure, such as the second generation vectors, to the
extent where, very often, the helper virus has a selective
advantage (more rapid replication).
[0012] The unresolved problem to this day of the production of
recombinant adenoviral vector particles with a high titer is an
obstacle to the development of gene therapy.
[0013] A novel helper virus has now been constructed by insertion
of direct repetitions on both sides of the encapsidation region.
The action of a recombinase recognizing them involves the excision
of the genetic material situated between them. This deletion does
not have any notable consequence on the expression of the viral
genes but limits the encapsidation of the helper virus in a viral
particle. Thus, the employment of the two-component procedure
described above in a cell line expressing the recombinase will
allow preparations enriched in adenoviral vector particles of
interest to be produced.
[0014] The present invention follows from the perfection of a
genetic technique based on the use of recombination sequences and
of a recombinase to produce mainly the recombinant viral vector and
to limit the contamination by the helper virus. The aim of the
present invention is to put at the disposal of the public a novel
helper virus able to express the genes which it carries (that is to
say capable of exercising its function of trans-complementation)
but unable to be propagated in the presence of a recombinase. The
solution provided by the present invention combines safety of use
(preparation enriched in recombinant viral vector), simplicity
(production in a conventional cell line in the presence of
recombinase) and efficiency (high titer compatible with industrial
needs). It is very particularly adapted to the production of second
generation adenoviral vectors.
[0015] For this reason the present invention relates to a helper
virus for the production of a recombinant viral vector defective
for replication, characterized in that it comprises a first
recombination sequence at 5' and a second recombination sequence at
3' of a region essential for the propagation of said helper virus;
said recombination sequences being recognized by a recombinase.
[0016] The term "helper virus" designates a vector able to
trans-complement in full or in part a recombinant viral vector
defective for replication. It is thus able to produce at least one
polypeptide, early and/or late, which the recombinant vector cannot
produce itself and which is necessary for the formation of a viral
particle. "In full" signifies that the helper virus is capable of
complementing the whole of the viral genome essential for
replication of which the recombinant viral vector is devoid and "in
part" signifies that the complementation is limited to a part of
the defective functions.
[0017] In the context of the present invention, a helper virus
derives from a natural virus such as found in nature as well as
from a virus whose genome comprises modifications with respect to
that of the parent virus from which it is descended. These
modifications can have been introduced in vitro by genetic
engineering techniques. They can be different (deletion, mutation
and/or addition of one or more nucleotides) and localized in the
encoding regions of the viral genome or outside of these. The
modification can, for example, allow one or more gene(s) essential
for viral replication to be inactivated with the aim of likewise
rendering it defective that is to say incapable of autonomous
replication.
[0018] The human adenoviruses of serotype C and, more particularly,
of type 2, 5 or 7 represent particularly preferred viruses in the
context of the invention. However, it is likewise possible to
resort to other adenoviruses, especially of animal origin (canine,
bovine, murine, avian, ovine, porcine or simian). It is more
particularly possible to mention the canine adenoviruses CAV-1 or
CAV-2, avian viruses DAV or even bovine viruses Bad of type 3
(Zakharchuk et al., 1993, Arch. Virol., 128, 171-176; Spibey and
Cavanagh, 1989, J. Gen. Virol., 70, 165-172; Jouvenne et al., 1987,
Gene, 60, 21-28; Mittal et al., 1995, J. Gen. Virol., 76, 93-102).
However, it may also be of interest to have a helper virus derived
from a poxvirus (vaccinia virus, fowlpox, canarypox . . . ),
retrovirus, herpesvirus, cytomegalovirus, adenovirus-associated
virus (AAV) or even at a hybrid virus comprising fragments of
different origin.
[0019] The characteristic of the helper virus according to the
invention is that it comprises at least two recombination sequences
inserted at 5' and at 3' of a sequence essential for its
propagation. An essential sequence designates all or part of a
viral gene, elements necessary for the expression such as a
promoter or, in a preferred manner, elements acting in cis (ITR,
LTR, encapsidation sequence . . . ). By way of information, the
first and/or second recombination sequences can be positioned in
the interior or immediately at 5' and 3' of the essential region up
to about several hundreds of bp.
[0020] In the sense of the present invention, a "recombination
sequence" is formed by a nucleic acid sequence (DNA or RNA)
recognized by a recombinase able to induce a recombination event.
Usually, a recombination sequence has at least 10 base pairs (bp),
advantageously 15 to 80 bp, preferably 20 to 60 bp and, in a very
preferred manner, 30 to 50 bp. According to an advantageous
embodiment, a helper virus according to the invention comprises two
copies of an identical or closely similar recombination sequence
(at least 70% sequence identity and, in a preferred manner at least
90%). For this reason we shall talk of sequence repetitions. In
this respect, the repetition can be reversed (the two recombination
sequences present in the helper virus have a reverse orientation
with respect to one another, one being in the direction 5' to 3'
and the other 3' to 5') or direct (same orientation 5' to 3' or 3'
to 5'). This second form will be preferred.
[0021] The recombination is accompanied by a pairing of the
recombination sequences, by a cleavage of a target sequence at
their level and by a ligation of the cleaved ends. The enzyme able
to promote the recombination is designated "recombinase". The
recombination between direct repetitions leads to the excision of
sequences between them. On the other hand, recombination between
reversed repetitions involves the reversal of the genetic material
located between them.
[0022] Generally speaking, the recombination sequences and the
recombinases are described in the literature accessible to the
person skilled in the art. They can be of any viral, phagic,
prokaryotic or eukaryotic origin (yeast, fungus or even higher
eukaryote). In addition, they can be obtained by the conventional
molecular biology techniques (cloning, amplification by chain
reaction (PCR for Polymerase Chain Reaction) or by chemical
synthesis.
[0023] As preferred examples, mention will be made of loxP
recombination sequences (described in the identifier of sequence
SEQ ID NO.: 1), FRT (SEQ ID NO.: 2) and R (SEQ ID NO.: 3)
recognized by the recombinases CRE, FLP and R, respectively (see,
for example, the review article Kilby et al., 1993, TIG 9,
413-420).
[0024] A helper virus particularly adapted to the present invention
is derived from the genome of an adenovirus and comprises the ITRs
5' and 3', an encapsidation region and at least one viral gene
selected from the genes of the E1, E2, E4 and L1-L5 regions and
defective in the recombinant adenoviral vector as well as a first
recombination sequence at 5' and a second recombination sequence at
3' of the encapsidation region. Preferably, they are formed by loxP
sequences positioned in the same orientation with respect to one
another. According to a first variant of interest, the
encapsidation region can be attenuated (reduced capacity for
encapsidation) to favor the encapsidation of the recombinant viral
vector. The attenuation can be obtained by deletion of a part of
the encapsidation region. The means of attenuating an encapsidation
region are indicated in the Application WO 94/28152.
[0025] According to a second variant of interest, the helper virus
can include an expression cassette of a recombinase, and allowing
especially an inducible expression or the production of an inactive
recombinase activatable according to needs (defined below). The
insertion takes place in an appropriate region of the helper virus
and, preferably, outside of the localized region between the
recombination sequences.
[0026] According to a particular embodiment intended to increase
the safety aspect, a helper virus according to the invention can
comprise a first recombination sequence and a second recombination
sequence at 5' and at 3' of several regions essential to its
propagation. For reasons of simplicity of employment, the case
will,be preferred where the recombination sequences are identical
or related so as to be recognized by an identical recombinase.
[0027] The present invention likewise relates to a complementation
cell line comprising a DNA fragment coding for a recombinase. It
can be generated from various cell lines by introduction of
appropriate portions of the viral genome and of the fragment in
question. A line able to complement the E1 and/or E4 function of an
adenovirus is more particularly preferred. Mention will be made of
the lines 293 (Graham, 1997 supra) and 1653 (described in the
Application WO 94/28152) modified by the introduction of the DNA
fragment coding for a recombinase.
[0028] All the standard means for introducing a nucleic acid into a
cell can be employed in the context of the present invention
(synthetic, viral, plasmid vector, naked DNA . . . ). Of course,
said DNA fragment can be integrated into the cell genome or remain
in the episome state. For the aims of the present invention, it can
comprise the elements necessary for its expression. These will be,
preferably, elements conferring an inducible expression in response
to an inducer. Such elements are known to the person skilled in the
art. It is possible to mention, by way of information, promoters
inducible by metals (promoter of the metallothioneine gene), by
hormones (promoter comprising elements responding to
glucocorticoids GRE, to progesterones PRE, to estrogens ERE . . .
), by viral inducers (promoter comprising the TRA or RRE sequence
responding respectively to the TAT or REV protein of the human
immunodeficiency virus HIV) or by various cellular inducers
(promoter comprising the UAS-Gal4 sequence (for Upstream Activating
Sequence Gal4) responding to Gal4 or the operators of the
tetracycline bacterial operon responding positively to the
trans-tetracycline activator tTA.
[0029] In addition, a DNA fragment-in use in the present invention
codes for a recombinase able to recognize the recombination
sequences present in a helper virus according to the invention. It
is preferred to employ a recombinase selected from the group formed
by CRE, FLP and R. However, it is equally possible to resort to a
DNA fragment coding for a homolog of a recombinase whose sequence
is modified with respect to the native sequence but exercising a
similar or improved function. These modifications can result from
the deletion, addition or substitution of one or more
nucleotide(s). They can likewise be a hybrid protein resulting from
the fusion of polypeptides from various origins, especially a
polypeptide having a recombinase activity and the other a linking
region. A recombinase particularly adapted to the present invention
is formed by a hybrid protein, designated CRE-ER, resulting from
the fusion of the recombinase CRE and of the linking region to the
ligand of the human estrogen receptor; (Metzger et al., 1995, Proc.
Natl. Acad. Sci. USA 92). The latter insofar as such is inactive
and its biological activity is activated in the presence of a
hormonal ligand such as estradiol.
[0030] The invention likewise relates to a process for preparing a
viral particle comprising a recombinant viral vector, which
comprises the following steps:
[0031] (a) Preparing a recombinant viral vector deficient for
replication;
[0032] (b) Preparing a helper virus according to the invention;
[0033] (c) Introducing the recombinant viral vector and the helper
virus into an appropriate cell line;
[0034] (d) Culturing said cell line under appropriate conditions to
allow the production of the viral particle in the presence of a
functional recombinase able to recognize said first and second
recombination sequences; and
[0035] (e) Recovering the viral particle in the cell culture.
[0036] In the sense of the present invention, a defective
recombinant viral vector derives from a virus in the genome of
which certain sequences have been deleted, rendered nonfunctional,
mutated or even substituted by other sequences and, more
particularly, a heterologous DNA fragment (normally not present in
the parent virus). The insertion takes place in an appropriate
region of the viral genome, so as to allow its expression in a host
cell. A host cell is formed by any eukaryotic cell infectable by a
viral particle containing said recombinant viral vector.
[0037] The heterologous DNA fragment in use in the present
invention can be descended from a eukaryotic organism, from a
prokaryote or from a virus other than that in which it is inserted.
It can be isolated by any technique conventional in the field of
the art, for example by cloning, PCR or chemical synthesis. It can
be a fragment of genome type (comprising all or part of the whole
of the introns), of complementary DNA type (cDNA, devoid of intron)
or of mixed type (comprising all or part of at least one intron).
In addition, it can code for an antisense RNA and/or a messenger
RNA (mRNA) which will then be translated into a polypeptide of
interest, the latter being able to be (i) intracellular, (ii)
membranous present at the surface of the host cell or (iii)
secreted into the external medium. In addition, it can be a
polypeptide as found in nature (native) or a portion of the latter
(truncated) or equally a chimeric polypeptide arising from the
fusion of sequences of various origins or even mutated and having
improved or modified biological properties.
[0038] In the context of the present invention, it can be
advantageous to use a DNA fragment coding for a cytokine
(interleukin including IL-2, interferon, colony-stimulating factor
. . . ), a cell or nuclear receptor, a ligand, a clotting factor
(factor VII, factor VIII, factor IX . . . ), CFTR protein (Cystic
Fibrosis Trans-membrane Conductance Regulator), insulin,
dystrophin, a growth hormone, an enzyme (renin urease, thrombin . .
. ), an enzyme inhibitor (inhibitor of a viral protease,
.alpha.1-antitryspin . . . ), a polypeptide with antitumor effect
(product of suppressor genes of tumors, polypeptide stimulating the
immune system . . . ), a polypeptide able to inhibit or slow down
the development of a bacterial, viral or parasitic infection
(antigenic polypeptide, trans-dominant variant . . . ) an antibody,
a toxin, an immunotoxin and finally a label (luciferase,
.beta.-galactosidase, product conferring resistance to an
antibiotic . . . ). Of course, this list is not limiting and other
genes can likewise be employed.
[0039] Advantageously, the heterologous DNA fragment is placed
under the control of elements necessary for its expression in the
host cell. "Necessary elements" designates all of the elements
allowing the transcription of said DNA fragment to RNA (antisense
RNA or mRNA) and the translation of the mRNA to polypeptide. These
elements comprise a regulatable or constitutive promoter, which can
be heterologous or on the contrary homologous to the parent virus.
It is possible to mention, as examples, the promoter of the human
or murine PGK gene (Phospho Glycerate Kinase), the early promoter
of the SV40 virus (Simian Virus), the LTR of RSV (Rous Sarcoma
Virus), the TK promoter (Thymidine Kinase) of the HSV-1 virus
(Herpes Simplex Virus) and the adenoviral promoters E1A and MLP.
The necessary elements can, in addition, include additional
elements (intron sequence, secretion signal sequence, nuclear
localization sequence, translation initiation site, transcription
termination poly A signal . . . ). Although it is not a preferred
variant, it is indicated that the viral vector can likewise
comprise a DNA fragment coding for the recombinase.
[0040] It is within reach of the person skilled in the art to
generate a recombinant viral vector in use in the present
invention. He will quite certainly know how to adapt the technology
as a function of the specific data (type of vector, heterologous
DNA fragment . . . ). According to a preferred variant, the vectors
capable of being employed in the context of the present invention
are recombinant adenoviral vectors defective for all of the viral
functions or even all of the functions with the exception of E4.
Such vectors are described in the International Application WO
94/28152.
[0041] A helper virus according to the invention is obtained by
insertion in a viral genome of a first and a second recombination
sequence on both sides of a region essential for replication and,
preferably, of the encapsidation region, it being possible for the
latter to be attenuated or nonattenuated. The person skilled in the
art knows the regions essential for the replication of a virus and
is able to carry out such a construction by applying the classical
techniques of molecular biology. According to the variant mentioned
above, it can likewise comprise a DNA fragment coding for a
recombinase and, especially, the CRE-ER hybrid. According to a
preferential embodiment, a helper virus according to the invention
and the recombinant viral vector which it allows production of
derive from the same parent virus and, in a very preferred manner,
from an adenovirus.
[0042] After the actual construction step, the helper virus and the
recombinant viral vector are introduced into an appropriate cell
line. All the standard means for introducing a nucleic acid into a
cell can be used in the context of the present invention, for
example transfection, electroporation, microinjection, lipofection,
adsorption and fusion of protoplasts. It is indicated that they can
be co-introduced (concomitant fashion) or introduced separately
(the helper virus according to the invention previously or
subsequently to the recombinant viral vector).
[0043] Although any cell line can be employed in the context of the
present invention, a complementation line is especially preferred.
Recourse will be had to a line of the prior art (293, 1653 . . . )
when a recombinant viral vector or a helper virus comprising the
DNA fragment coding for the recombinase is employed. On the other
hand, when this is not the case, use will be made of a
complementation cell line according to the invention.
[0044] After transfection, the cell line is cultured under
appropriate conditions to allow the production of viral particles.
A procedure according to the invention can, in addition, comprise
an amplification step previous to the culture step in the presence
of the functional recombinase. The aim of this step is to increase
the quantities of helper virus and of recombinant viral vector in
order to improve the yields. It can be carried out by culture in
any permissive line or in the appropriate line in use in the
present invention before the addition, expression or activation of
the recombinase.
[0045] This first culture step is followed by a second step carried
out in the presence of a functional recombinase able to recognize
said first and second recombination sequences. In the context of
the procedure according to the invention, this recombinase can be
added to the cell culture, for example in substantially pure form.
However, according to another very preferred and already mentioned
variant, the recombinase is produced by one of the constituents of
the procedure according to the invention, namely the recombinant
viral vector or, in a preferred manner, the helper virus or the
cell line. Once produced in functional form, the recombinase will
cause the excision of the essential region of the helper virus
localized between the recombination sequences, with the aim of
preventing or reducing its propagation.
[0046] In addition, when a recombinase is employed whose expression
is inducible by an inducer or the CRE-ER hybrid protein whose
biological activity is dependent on a hormonal ligand, the culture
step in the presence of the functional recombinase is carried out
by the addition to the culture medium of the inducer or the
ligand.
[0047] According to an advantageous embodiment intended to increase
the safety of a procedure according to the invention, the helper
virus and the recombinant viral vector are defective and can be
conversely complemented, in total or in part. A variant of interest
consists in employing (i) a helper adenovirus according to the
invention defective for the functions E1 and E4 and comprising the
loxP sequences at 5' and at 3' of the encapsidation region,
(attenuated or nonattenuated) (ii) a recombinant vector defective
for all the functions with the exception of E4 and (iii) a 293 cell
line producing the CRE-ER hybrid recombinase. According to another
advantageous alternative, a procedure according to the invention
employs (i) a helper adenovirus according to the invention
defective for the functions E1 and E4, comprising the loxP
sequences at 5' and at 3' of the encapsidation region (attenuated
or nonattenuated) and producing the CRE-ER hybrid recombinase, (ii)
a recombinant vector defective for all of the functions with the
exception of E4 and (iii) a conventional cell line 293.
[0048] The viral particles are recovered from the cell culture,
from the medium or after lysis of the cells. Advantageously, a
procedure according to the invention comprises an additional step
of purification of the recombinant viral vector particles. Although
the choice of the technique is wide and within the reach of the
person skilled in the art, it is possible to mention more
particularly ultracentrifugation on a cesium chloride or sucrose
gradient.
[0049] Finally, the invention likewise relates to a procedure for
preparing a viral particle comprising a recombinant viral vector by
means of a helper virus, according to which the ratio viral
particles of recombinant adenoviral vector to those of helper virus
is greater than 50%, advantageously greater than 60%, preferably
greater than 70% and, in a very preferred manner, greater than
80%.
[0050] The invention likewise relates to a recombinant viral vector
particle obtained by a procedure according to the invention as well
as to a eukaryotic host cell according to the invention. Said host
cell is advantageously a mammalian cell and, preferably, a human
cell and can comprise said vector in integrated form in the genome
or in nonintegrated form (episome). It can be a primary or tumor
cell of hematopoietic (totipotent stem cell, leukocyte, lymphocyte,
monocyte or macrophage . . . ), muscular, pulmonary, tracheal,
hepatic, epithelial or fibroblast origin.
[0051] The invention likewise relates to a pharmaceutical
composition comprising as therapeutic or prophylactic agent a
recombinant viral vector particle obtained by a procedure according
to the invention or a eukaryotic host cell according to the
invention, in combination with a carrier acceptable from a
pharmaceutical point of view. The composition according to the
invention is intended in particular for the preventive or curative
treatment of diseases such as:
[0052] genetic diseases (hemophilia, mucoviscidosis, diabetes or
myopathy, that of Duchene and Becker . . . ),
[0053] cancers, such as those induced by oncogenes or viruses,
[0054] viral diseases, such as hepatitis B or C or AIDS (acquired
immunodeficiency syndrome resulting from infection by HIV), and
[0055] recurrent viral diseases, such as viral infections caused by
the herpesvirus.
[0056] A pharmaceutical composition according to the invention can
be produced in a conventional manner. In particular, a
therapeutically efficacious quantity of a therapeutic or
prophylactic agent is combined with a carrier such as a diluent. A
composition according to the invention can be administered by
aerosol, locally or even systemically. The routes of administration
envisagable within the context of the present invention can be
intragastric, subcutaneous, intracardiac, intramuscular,
intravenous, intraperitoneal, intratumor, intrapulmonary, nasal or
intratracheal. The administration can take place in a single or
repeated dose one or more times after a certain delay interval. The
appropriate route of administration and the appropriate dose vary
as a function of various parameters, for example of the individual
or of the disease to be treated or even of the recombinant gene(s)
to be transferred. In particular, the viral particles according to
the invention can be formulated in the form of doses of between
10.sup.4 and 10.sup.14 pfu (plaque-forming units), advantageously
10.sup.5 and 10.sup.13 pfu and, preferably, 10.sup.6 and 10.sup.11
pfu. The formulation can likewise include an adjuvant which is
acceptable from a pharmaceutical point of view.
[0057] Finally, the present invention relates to the therapeutic or
prophylactic use of a recombinant viral vector particle obtained by
a procedure according to the invention or of a eukaryotic host cell
according to the invention for the preparation of a medicament
intended for the treatment of the human or animal body and,
preferentially, by gene therapy. According to a first possibility,
the medicament can be administered directly in vivo (for example in
an accessible tumor, in the lungs by aerosol . . . ). It is
likewise possible to adopt the ex vivo approach which consists in
taking cells from the patient (stem cells of the bone marrow,
peripheral blood lymphocytes, muscle cells . . . ), in transfecting
or infecting them in vitro according to the techniques of the art
and in readministering them to the patient.
[0058] The invention likewise extends to a method of treatment
according to which a therapeutically efficacious quantity of a
recombinant viral vector particle obtained by a procedure according
to the invention or of a eukaryotic host cell according to the
invention is administered to a patient having need of such a
treatment.
[0059] The present invention is described more completely with
reference to the following figures and with the aid of the
following examples.
[0060] FIG. 1 is a schematic representation of the genome of the
human adenovirus of type 5 (represented in arbitrary units from 0
to 100), indicating the position of the different genes.
[0061] FIG. 2 is a schematic representation of the pTG4701 vector
in which the left 5' region of the 5 adenovirus is modified by the
insertion of LoxP direct repetitions on both sides (in positions
161 and 460) of the encapsidation region (psi).
[0062] FIG. 3 is a schematic representation of the pTG8595 vector
with the E1, E3 and E4 regions and comprising an expression
cassette of the LacZ gene controlled by the MLP promoter and a
polyA sequence (pA).
[0063] FIG. 4 is a schematic representation of the pTG4707 vector
with the E1, E3 and E4 regions and comprising two LoxP direct
repetitions on both sides of the psi encapsidation region.
[0064] FIG. 5 is a schematic representation of the pTG4662 vector,
a recombinant adenoviral vector with the E1 and E3 regions and
carrying the same LacZ cassette as pTG8595.
[0065] FIG. 6 is a schematic representation of the vector pTG4667,
a recombinant adenoviral vector with the E1, E3 regions and the
ORFs 1 to 4 of E4 deleted and carrying the same LacZ cassette as
pTG8595.
[0066] FIG. 7 is a schematic representation of the pTG4702 vector
which derives from the above vector by insertion of an expression
cassette of the CRE-ER hybrid protein (indicated cre and hER) in
place of that of LacZ.
EXAMPLES
[0067] The following examples only illustrate one method of
carrying out the present invention.
[0068] The constructs described below are carried out according to
the general techniques of gene therapy and of molecular cloning,
detailed in Maniatis et al., (1989, Laboratory Manual, Cold Spring
Harbor, Laboratory Press, Cold Spring Harbor, N.Y.) or according to
the recommendations of the manufacturer when using a commercial
kit. The cloning steps employing bacterial plasmids are carried out
in the strain Escherichia coli (E. coli) 5K (Hubacek and Glover,
1970, J. Mol. Biol. 50, 111-127) or BJ5183 (Hanahan, 1983, J. Mol.
Biol. 166, 557-580). This latter strain is preferentially used for
the homologous recombination steps. The techniques of amplification
by PCR are known to the person skilled in the art (see, for
example, PCR Protocols--A guide to methods and applications, 1990,
edited by Innis, Gelfand, Sninsky and White, Academic Press Inc.).
Being a question of the repair of restriction sites, the technique
employed consists in filling the protruding 5' ends with the aid of
the large fragment of DNA polymerase I from E. coli (Klenow).
[0069] As far as the cellular biology is concerned, the cells are
transfected according to the standard techniques well known to the
person skilled in the art. The calcium phosphate technique can be
mentioned (Maniatis et al., supra), but any other protocol can
likewise be employed, such as the DEAE dextran technique,
electroporation, methods based on osmotic shock, microinjection of
a selected cell or methods based on the use of liposomes. As for
the culture conditions, they are conventional except when
specified.
[0070] In the examples which follow, recourse is had to the
following cell lines:
[0071] Line 293 derived from embryonic human kidney (Graham et al.,
1977, supra) which results from the integration into its
chromosomes of the 5' end of the genome of Ad5 (ITR5',
encapsidation sequence and E1 region) (available at the ATCC under
reference 1573).
[0072] Line TG1653 (described in the International Application
WO94/28152, Example 8) which derives from the line 293 transformed
in a stable manner by the plasmid pTG1653 carrying the E4 region of
Ad5 (nt 32800 to 35826) and the expression cassette of the pac
(Puromycin Acetyl Transferase) puromycin resistance gene
(Morgenstern and Land, 1990, Nucleic Acids Res. 18, 3587-3596).
[0073] It is understood that other cell lines can be used.
[0074] In addition, the fragments of adenoviral genome employed in
the different constructs described below are indicated precisely
according to their positions in the nucleotide sequence of the
genome of Ads such as disclosed in the Genebank databank under the
reference M73260.
Example 1
Formation of a Helper Virus
[0075] This example describes the construction of an adenoviral
helper virus defective for the functions E1 and E4 and comprising
an loxP recombination sequence positioned on both sides of the
encapsidation region.
[0076] The loxP sequence is carried by the oligonucleotides
nucleotides oTG6374 and oTG6522 (SEQ ID NO: 4 and 5). These are
reassociated and then introduced into the HindIII site of the p
poly II vector (Lathe et al., Gene 57, 193-201) to give
pTG4691.
[0077] The vector pTG8343 arises from the insertion in the p poly
II vector of a part of the 5' end of the adenoviral genome, namely
the sequences extending from the nucleotides (nt) 1 to 458 and 3328
to 5788. This is linearized by SalI and ligated with the SalI-XhoI
fragment isolated from pTG4691 carrying the loxP sequence. pTG4695
is obtained, which comprises an loxP sequence in position 450 of
the adenoviral genome or at 3' of the encapsidation region. A
second loxP sequence is cloned in the form of a SmaI-PvuII fragment
purified of pTG4691 in the preceding vector linearized by AflII
(position 161) and treated with Klenow. The pTG4701 vector thus
obtained comprises two direct loxP repetitions surrounding the psi
encapsidation region (FIG. 2).
[0078] The modified encapsidation region is exchanged for its
genomic homolog by the technique of homologous recombination. To
this end, the E. coli strain is co-transformed with the BglI
fragment of pTG4701 and the pTG8595 vector (FIG. 3) digested by
ClaI. The latter is a recombinant adenoviral vector with the E1 (nt
459-3328), E3 (nt 28592-30470) and E4 (nt 32800-35826) regions
deleted and comprising an expression cassette of the LacZ gene
under the control of the MLP promoter in place of the E1 region.
The recombined vector is designated pTG4707 (FIG. 4). After
transfection in the TG1653 complementation line (E1.sup.+,
E4.sup.+) and amplification, the Ad TG4707 viruses are purified on
a cesium chloride gradient so as to form a viral stock titrating
approximately 2.times.10.sup.8 pfu/ml.
Example 2
Construction of a Recombinant Adenoviral Vector Producing a
Recombinase
[0079] This example describes the construction of an adenoviral
vector expressing the CRE-ER hybrid protein. In the absence of
estrogen, the latter is produced in an inactive form but in the
presence of the hormone, it adopts an active conformation. The
adenoviral vector is devoid of most of the E1 and E3 regions and of
the part of the E4 region other than the ORFs 6 and 7 (Open Reading
Frame). The expression of these two genes is sufficient to assure
the E4 function without necessity for complementation (Ketner et
al., 1989, Nucleic Acids Res. 17, 3037-3048). Two types of vectors
have been constructed. In the first (pTG4708), the CRE-ER gene is
placed under the control of the SV40 promoter although in the
second (pTG5630), it is directed by the CMV promoter.
[0080] In the first place, the pTG1653 vector carrying the E4
region of Ad5 (nt 32800 to 35826; see WO 94/28152), is digested by
AvrII and BglII and then treated by Klenow before being religated
on itself. pTG4660 which carries an expression cassette of the ORFs
6 and 7 under the control of the homologous promoter E4 is
obtained. This is introduced into an adenoviral vector by
homologous recombination. To this end, the vector pTG4662 (FIG. 5)
is chosen which comprises the ITR 5' and the encapsidation
sequences (nt 1 to 458), an expression cassette of the LacZ gene in
the place of the E1 region and the remaining adenoviral sequences
with the E3 region (nt 3329 to 27870 and 30749 to 35935) deleted.
The E. coil BJ strain is co-transformed by the FspI-MunI fragment
descended from pTG4660 and pTG4662 digested by SwaI. This
recombination event allows pTG4667 (FIG. 6) to be generated, in
which the cassette ORFs 6 and 7 replace the E4 wild region.
[0081] A. Construction of the pTG4708 Vector
[0082] In parallel, the expression cassette of the CRE-ER hybrid
recombinase is isolated from the pCre-ER vector (Metzger et al.,
1995, supra) in the form of a SalI fragment and cloned in the
vector pTGB343 previously linearized by this same enzyme. pTG4699
and pTG4700 which differ by the orientation of the cassette are
obtained.
[0083] The vectors pTG4702 (FIG. 7) and pTG4703 are obtained by
homologous recombination between a purified SgrAI-BstEII fragment
of pTG4699 and pTG4700 respectively and the adenoviral vector
pTG4667 linearized by ClaI. They carry the CRE-ER expression
cassette (different orientation for each of them) in place of the
E1 region and are devoid of the E3 region and of the ORFs 1 to 4 of
E4. They are transfected in the 293 and 1653 cells so as to ensure
that the expression of the recombinase is not toxic to cell growth
and to viral multiplication.
[0084] The construction of an adenoviral vector, in addition,
defective for the E2 region can be carried out starting from
previous constructs by treatment with the AscI enzyme and
religation. The clones are isolated which have reintegrated the
AscI fragment but in opposite orientation with respect to the
parent vector. The candidates can be determined by simple enzymatic
digestion with enzymes whose sites are present on the fragment, for
example BamHI and are designated pTG4708. The aim of this reversal
of the orientation is to interrupt the transcription units coding
for E2A, the polymerase and the hexon and to render the E2 function
defective. It is equally possible to introduce deletions of all or
part of the E2 region.
[0085] B. Construction of the pTG5630 Vector
[0086] The vector pTG4667 is digested by the SnaBI enzyme and then
religated. The vector pTG5613 similar to pTG4667 is selected except
for the reversal of the adenoviral fragment SnaBI (positions 10307
to 25173) covering a large part of the coding sequences of the E2
region. This reversal allows a defective virus to be generated
which is incapable of producing the functional expression products
of E2.
[0087] In addition, the sequences coding for CRE-ER are isolated by
EcoRI digestion and treated by Klenow before being introduced
upstream of the CMV promoter, giving rise to pTG5625. The vector
pTG5630 is obtained by homologous recombination between the
PacI-BstEII fragment isolated from pTG5625 and the vector pTG5617
linearized by ClaI. pTG5630 is defective for the E1 and E2
functions, with the major part of E3 deleted, and carries the
"CMV--CRE-ER promoter" cassette in place of E1 in reverse
orientation with respect to the 5' ITR.
Example 3
Procedure for Preparation of Viral Particles of an Adenoviral
Vector by the System CRE and loxP
[0088] A. Employment of the Vector pTG4708 and the Helper
pTG4707
[0089] In this example, the following are used:
[0090] (1) an adenoviral vector (pTG4708) defective for all of the
adenoviral functions with the exception of E4 and carrying the
sequences coding for the recombinase CRE-ER,
[0091] (2) a helper virus (pTG4707) defective for the functions E1,
E3 and E4 and containing two loxP direct repetitions placed on both
sides of the encapsidation region, and
[0092] (3) the 293 complementation cell line complementing the E1
function.
[0093] The vectors pTG4707 and pTG4708 are co-transfected in 293
cells and cultured at first in a conventional medium not containing
estrogen. The viral particles can be formed in the cells which
comprise the two vectors since they are mutually complementary. In
fact, the E2 proteins are produced from the helper virus, the E4
proteins from the adenoviral vector and the E1 proteins are
supplied by the cell line. In addition, the CRE-ER hybrid protein
is produced in its inactive form since the culture medium is not
supplemented by hormone such that the genome of the helper virus is
able to be encapsidated. The plaques thus produced contain a mixed
population of virus, one part containing the genome of the
adenoviral vector and the other part that of the helper virus. This
step allows an amplification of the quantity of virus with the aim
of improving the titers.
[0094] The step of selective production of the particles of
adenoviral vector is carried out by introducing into the culture
medium estradiol according to the conditions detailed in Metzger et
al. (1995, supra) . The presence of estradiol will allow the CRE-ER
recombinase to be activated which, once functional, is able to
induce a recombination event between the loxP sequences. The
viruses generated under these conditions are purified, the DNA is
isolated and the deletion of the encapsidation region verified
either by enzymatic digestion or hybridization with an appropriate
probe complementary to that according to the technology of
Southern.
[0095] B. Employment of the Vector pTG5630 and of the Helper
pTG4707
[0096] 2.5 to 5 .mu.g of pTG5630 vector are transfected in the 293
line under the conventional conditions. The following day the
transfected cells are superinfected by the helper virus AdTG4707 at
a rate of approximately 0.04 pfu/cell and cultured in a DMEM medium
depleted of Phenol Red (the latter being capable of having an
estrogenic activity). Two conditions for culture were studied in
parallel: medium supplemented by .gamma.-estradiol (Sigma; E4389)
at a concentration of 10.sup.-6 and 10.sup.-7M after the third
passage and nonsupplemented medium. The cell culture is harvested
at 4 days post-infection and a part of the harvest amplified by
successive passages over fresh 293 cells. The other part is
preserved for the viral DNA analyses. A significant difference is
observed in the state of the cells according to the culture
conditions. In fact, the cytopathy is less pronounced in the
presence of estradiol than in its absence, this counting from the
third passage, which allows a difference to be shown at the level
of viral production.
[0097] In order to verify this point, the DNA of the viruses
produced during the four first passages is isolated from an
identical volume of culture, digested by the enzyme MunI and
analyzed by Southern with the aid of a radioactive probe
complementary to a sequence upstream of the E4 region. After
hybridization, an MunI fragment of 1.7 kb is visualized in the case
of the viruses descended from the pTG5630 vector and a MunI
fragment of 1.1 kb in the case of the AdTG4707 helper viruses.
These conditions allow the relative quantity of virus of interest
and helpers generated in each amplification cycle to be seen.
[0098] Under the conditions where the CRE-ER recombinase is
inactive (in the absence of estrogen), a simultaneous amplification
of the two types of virus is observed showing itself by a more and
more intense signal in proportion to the passages. On the other
hand, when the culture medium is supplemented by estradiol, the
signal corresponding to the helper virus decreases although that
specific for AdTG5630 increases with each passage. These results
indicate that the activation of the recombinase is accompanied by a
preferential amplification of the virus of interest.
[0099] The excision of the encapsidation region of the helper virus
is demonstrated by Southern analysis on the viral DNA preparations
digested by the enzyme AflII and employing a specific probe of the
encapsidation region hybridizing to a fragment of 3.7 kb in the
case of the AdTG5630 virus and to a fragment of either 800 bp in
the case of the complete helper virus or 400 bp in the case where
the encapsidation sequences bordered by the loxP sites are
excised.
[0100] The intensity of the signal corresponding to the fragment of
3.7 kb increases in proportion to amplification cycles whatever the
culture conditions although that corresponding to the 800 bp
fragment only increases in the absence of estradiol. On the other
hand, in the presence of the hormone, the signal corresponding to
the helper virus grows weaker. With high exposure, it is possible
to demonstrate a band at 400 pb indicating that the encapsidation
region of the helper viruses is excised by the action of the
recombinase.
[0101] In their entirety, these results show that the CRE-LoxP
system can be adapted to the adenoviruses to reduce the
contamination of the adenoviral preparations by the helper
virus.
Example 4
Formation of a Stable Line Expressing the CRE-ER Hybrid
Recombinase
[0102] The pCre-ER vector is transfected, in a conventional manner,
in the 293 cells at the same as a selection vector (for example
pRC-CMV conferring resistance to commercially available neomycin
(Invitrogen), pTG1643 conferring resistance to puromycin described
in WO 94/28156). After transfection, the cells are cultured in
selective medium containing the antibiotic and the resistant clones
(designated 293/CRE-ER) are isolated and are tested for their
capacity to produce an adenoviral vector.
[0103] The same technology is used to generate a 1653 line
(complementing the adenoviral functions E1 and E4) expressing the
CRE-ER gene product.
[0104] The 293/CRE-ER cells thus generated are co-transfected by
the pTG4707 helper virus and a recombinant adenoviral vector
defective for all the functions with the exception of E4 (such as
those described in WO 94/28152) and then cultured in a conventional
medium so as to generate a mixed and amplified population of viral
particles. After a certain time, the culture medium is replaced by
a medium containing estradiol in order to produce the recombinase
in its active form and to prevent the production of AdTG4707 viral
particles.
[0105] The 1653/CRE-ER line will be useful for the preparation of
vectors defective for all of the essential functions (see WO
94/28156). The technology employed is comparable to that described
above, namely: co-transfection fection by the pTG4707 helper virus
and a minimum recombinant adenoviral vector, culture in selective
medium which is not supplemented by estradiol for a sufficient time
and then addition of the hormone to the culture medium and recovery
of the viral particles produced.
Sequence CWU 1
1
5 1 34 DNA Bacteriophage P1 1 ataacttcgt ataatgtatg ctatacgaag ttat
34 2 34 DNA Saccharomyces cerevisiae 2 gaagttccta tactttctag
agaataggaa cttc 34 3 31 DNA Zygosaccharomyces rouxii 3 ttgatgaaag
aatacgttat tctttcatca a 31 4 45 DNA synthetic oligonucleotide
(loxP) 4 agctataact tcgtataatg tatgctatac gaagttatct cgaga 45 5 45
DNA synthetic oligonucleotide (loxP) 5 agcttctcga gataacttcg
tatagcatac attatacgaa gttat 45
* * * * *