U.S. patent application number 10/141491 was filed with the patent office on 2003-01-30 for method for producing recombinant virus.
Invention is credited to Leblois-Prehaud, Helene, Perricaudet, Michel, Vigne, Emmanuelle, Yeh, Patrice.
Application Number | 20030022356 10/141491 |
Document ID | / |
Family ID | 9497899 |
Filed Date | 2003-01-30 |
United States Patent
Application |
20030022356 |
Kind Code |
A1 |
Leblois-Prehaud, Helene ; et
al. |
January 30, 2003 |
Method for producing recombinant virus
Abstract
The invention concerns a method for producing recombinant virus.
This method is based on the use of baculovirus for providing the
complementary functions. It also concerns constructs used for
implementing this method, the producing cells, and the resulting
virus.
Inventors: |
Leblois-Prehaud, Helene;
(Guyancourt, FR) ; Perricaudet, Michel; (Ecrosnes,
FR) ; Vigne, Emmanuelle; (Ivry Sur Seine, FR)
; Yeh, Patrice; (Gif Sur Yvette, FR) |
Correspondence
Address: |
WILEY, REIN & FIELDING, LLP
ATTN: PATENT ADMINISTRATION
1776 K. STREET N.W.
WASHINGTON
DC
20006
US
|
Family ID: |
9497899 |
Appl. No.: |
10/141491 |
Filed: |
May 9, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10141491 |
May 9, 2002 |
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09308398 |
May 18, 1999 |
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6387670 |
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09308398 |
May 18, 1999 |
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PCT/FR97/02073 |
Nov 18, 1997 |
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Current U.S.
Class: |
435/235.1 ;
435/456 |
Current CPC
Class: |
C12N 2710/14143
20130101; C12N 2710/14043 20130101; C12N 2710/10343 20130101; C12N
2830/002 20130101; C12N 15/86 20130101; C12N 2800/30 20130101; A61K
48/00 20130101 |
Class at
Publication: |
435/235.1 ;
435/456 |
International
Class: |
C12N 007/00; C12N
015/861 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 22, 1996 |
FR |
FR96/14278 |
Claims
1. Process for the production of defective recombinant viruses
according to which the genome of the defective recombinant virus
and a baculovirus comprising all or some of the functions necessary
for the transcomplementation of the defective recombinant genome
are introduced into a population of competent cells.
2. Process according to claim 1, characterized in that the
baculovirus comprises all the functions necessary for the
transcomplementation of the defective recombinant genome.
3. Process according to claim 1, characterized in that the
baculovirus comprises some of the functions necessary for the
transcomplementation of the defective recombinant genome, the rest
being provided by the competent cell.
4. Process according to claim 1, characterized in that the
functions necessary for the transcomplementation of the defective
recombinant genome are provided by several baculoviruses.
5. Process according to claim 1, characterized in that the
defective recombinant virus is a defective recombinant
adenovirus.
6. Process according to claim 5, characterized in that the genome
of the recombinant adenovirus is defective for one or more
functions chosen from E1, E2, E3, E4, L1-L5, pIX and IVa2 and the
baculovirus comprises all the functions necessary for the
transcomplementation of the defective recombinant genome.
7. Process according to claim 5, characterized in that the genome
of the recombinant adenovirus is defective for one or more
functions chosen from E1, E2, E3, E4, L1-L5, pIX and IVa2, the
baculovirus comprises some of the functions necessary for the
transcomplementation of the defective recombinant genome, the rest
of the functions being provided by one or more other baculoviruses
and/or by the competent cell.
8. Process according to claim 5, characterized in that the helper
baculovirus comprises all or part of the E1 region of the
adenovirus, allowing the complementation of a recombinant
adenovirus genome defective for the E1 region.
9. Process according to claim 5, characterized in that the helper
baculovirus comprises all or part of the E2 region of the
adenovirus, allowing the complementation of a recombinant
adenovirus genome defective for the E2 region.
10. Process according to claim 5, characterized in that the helper
baculovirus comprises all or part of the E4 region of the
adenovirus, allowing the complementation of a recombinant
adenovirus genome defective for the E4 region.
11. Process according to claim 5, characterized in that the helper
baculovirus comprises all or part of the E1 and E4 regions of the
adenovirus, allowing the complementation of a recombinant
adenovirus genome defective for the E1 and E4 regions.
12. Process according to claim 5, characterized in that the
recombinant adenovirus genome lacks any coding viral region and the
helper baculovirus comprises all the functions allowing its
complementation.
13. Process according to claim 12, characterized in that the
baculovirus comprises the whole of an adenoviral genome, with the
exception of the encapsidation region and possibly of the ITRs.
14. Process according to claim 1, characterized in that the
complementation functions present in the baculovirus and the genome
of the defective recombinant virus do not comprise a zone of
homology capable of giving rise to recombination.
15. Process according to claim 14, characterized in that a
recombinant adenovirus genome defective for the E1 and possibly E3
region is introduced into the competent cells, these cells are
infected, simultaneously or otherwise, with a baculovirus
comprising the E1 region, the adenovirus E1 region present in the
baculovirus and the genome of the defective recombinant adenovirus
comprising no zone of homology capable of giving rise to
recombination.
16. Process according to claim 15, characterized in that the
baculovirus comprises a fragment 391-3511 of the Ad5 adenovirus and
in that the genome of the recombinant adenovirus defective for the
E1 region carries a larger deletion.
17. Process according to claim 16, characterized in that the
baculovirus comprises a fragment 391-3511 of the Ad5 adenovirus and
in that the genome of the recombinant adenovirus defective for the
E1 region carries a deletion covering nucleotides 383-3512
inclusive.
18. Recombinant baculovirus comprising, inserted into its genome, a
nucleic acid encoding a complementation function of a defective
virus placed under the control of a heterologous promoter.
19. Baculovirus according to claim 18, characterized in that the
complementation function is chosen from all or some of the
functions encoded by the E1, E2, E4, L1-L5, pIX and IVa2 regions of
the adenovirus, alone or in combinations.
20. Baculovirus according to claim 18, characterized in that the
complementation function is chosen from all or some of the
functions encoded by the Rep and Cap regions of the AAV, alone or
in combinations.
21. Baculovirus according to claim 18, characterized in that the
complementation function is chosen from all or some of the
functions encoded by the gag, pol and env regions of a retrovirus,
alone or in combinations.
22. Baculovirus according to claim 18, characterized in that the
nucleic acid encoding the complementation function consists of a
DNA corresponding to a fragment of a genome of the virus comprising
the corresponding region.
23. Baculovirus according to claim 22, characterized in that the
nucleic acid encoding the complementation function consists of a
DNA corresponding to a fragment of a genome of serotype Ad2 or Ad5
adenovirus.
24. Baculovirus according to claim 18, characterized in that the
promoter consists of the promoter region naturally responsible for
the expression of the complementation functions.
25. Baculovirus according to claim 18, characterized in that the
promoter is a strong cellular or viral promoter, regulated or
otherwise.
26. Baculovirus according to claim 19, characterized in that the
complementation function comprises the E1 region of an adenoviral
genome or only a part thereof comprising at least the E1a
region.
27. Baculovirus according to claim 19, characterized in that the
complementation function comprises the E4 region of an adenoviral
genome or only a part thereof comprising at least the frame ORF3 or
ORF6.
28. Baculovirus according to claim 19, characterized in that it
comprises all the coding regions of an adenoviral genome.
29. Baculovirus according to claim 28, characterized in that it
comprises a complete adenoviral genome, lacking the encapsidation
region.
30. Baculovirus according to claim 18, characterized in that it is
an AcNPV strain.
31. Baculovirus according to claim 18, characterized in that the
nucleic acid is introduced at the level of the polyhedrin locus or
of the p10 locus.
32. Baculovirus according to claim 18, characterized in that the
nucleic acid is introduced in the form of a cassette which is
capable of being excised in the competent cell.
33. Recombinant baculovirus comprising, inserted into its genome,
at least one DNA region flanked by two sequences allowing a
site-specific recombination and positioned in direct orientation,
the said DNA region comprising at least one replications origin
functional in competent cells and a nucleic acid encoding a
complementation function of a virus.
34. Baculovirus according to claim 33, characterized in that the
sequences allowing a site-specific recombination are LoxP sequences
of the P1 bacteriophage, and the recombination is obtained in the
presence of the Cre protein.
35. Process according to claim 5, characterized in that the
defective recombinant genome is introduced into the cell by
infection with an adenovirus comprising the said genome.
36. Process according to claim 5, characterized in that the
defective recombinant genome is introduced into the cell by
transfection.
37. Process according to claim 1, characterized in that the
defective recombinant genome is introduced into the cell with a
recombinant baculovirus, distinct from the baculovirus carrying the
complementation functions.
38. Recombinant baculovirus comprising, inserted into its genome,
at least one DNA region flanked by two sequences allowing a
site-specific recombination and positioned in direct orientation,
the said DNA region comprising at least one replication origin
functional in competent cells and a defective adenovirus
genome.
39. Baculovirus according to claim 38, characterized in that the
defective recombinant adenovirus genome comprises essentially the
ITR regions, the encapsidation sequence and a nucleic acid of
interest.
40. Process for the production of defective recombinant
adenoviruses, characterized in that a population of competent cells
is infected with a baculovirus according to claim 33 and with a
baculovirus according to claim 38, the cells are put in the
presence of the recombinase allowing the site-specific
recombination, and then the adenoviruses produced are
recovered.
41. Process according to claim 1, characterized in that the
population of competent cells is a population of hepatic, muscle,
fibroblast, embryonic, epithelial (particularly pulmonary), ocular
(particularly retinal) or nerve cells.
42. Process according to claim 41, characterized in that the
population of competent cells is chosen from the cells 293 or any
derived cell comprising an additional complementation function,
A549, HuH7, Hep3B, HepG2, HER, 911, HeLa or KB.
43. Purified viral preparation obtained using the process according
to claim 1.
Description
[0001] The present invention relates to a method for the production
of recombinant viruses. It also relates to constructs used for
carrying out this method, the producing cells, and the viruses thus
produced. These viruses can be used as vector for the cloning
and/or expression of genes in vitro, ex vivo or in vivo.
[0002] Vectors of viral origin are widely used for the cloning,
transfer and expression of genes in vitro (for the production of
recombinant proteins, for carrying out screening tests, for
studying the regulation of genes and the like), ex vivo or in vivo
(for the creation of animal models, or in therapeutic approaches).
Among these viruses, there may be mentioned in particular
adenoviruses, adeno-associated viruses (AAV), retroviruses,
herpesviruses or vaccinia viruses.
[0003] The Adenoviridae family is widely distributed in mammals and
birds and comprises more than one hundred different serotypes of
nonenveloped double-stranded DNA viruses possessing a capsid of
icosahedral symmetry (Horwitz, In: Fields B N, Knipe D M, Howley P
M, ed. Virology. Third edition ed. Philadelphia: Raven Publishers,
1996: 2149-2171). In addition to its safety, the adenovirus has a
very broad cellular tropism. Unlike the retrovirus, whose cycle is
dependent on cell division, it can infect actively dividing cells
such as quiescent cells and its genome is maintained in episomal
form. Furthermore, it can be produced at high titres (10.sup.11
pfu/ml). These major assets of it one makes a most preferred vector
for the cloning and expression of heterologous genes. The group C
adenoviruses, particularly types 2 and 5, as well as the CAV-2-type
canine adenoviruses, whose molecular biology is best known, are the
source of the vectors currently used.
[0004] The adenovirus has a linear genome of 36 kb, terminating at
each of these ends with inverted terminal repeat (ITR) sequences of
103 bp comprising a replication origin as well as an encapsidation
signal situated near the left ITR (Shenk, Adenoviridae: The Viruses
and Their Replication. In: Fields B N, Knipe D M, Howley P M, ed.
Virology. Philadelphia: Raven publishers, 1996: 2111-2148). Three
families of genes are expressed during the viral cycle:
[0005] The immediate-early genes (E1, E2, E3 and E4) which are
involved in the regulation of cellular genes allowing in particular
the entry of the cell into the S phase (E1A) and the inhibition of
apoptosis (E1B). They are also involved in the regulation of early
or late viral genes at the level of the transcription, splicing or
transport of the messenger RNAs (E1A E2A, E4). They also play a
role in replication and in escaping the immune response.
[0006] The delayed-early genes (pIX and IVa2) are linked to the
regulation of transcription of the late genes (IVa2) or to the
assembling of the virion (pIX).
[0007] The late genes (L1 to L5) are transcribed from the strong
promoter (MLP). A primary transcript of 28 kb makes it possible to
generate the transcripts corresponding to the various structural
proteins (core, penton, hexon) and nonstructural proteins
participating in the assembling and in the maturation of the viral
particles, by alternative splicing and the use of 5 polyadenylation
sites.
[0008] Adenoviral vectors have been used for the cloning and
expression of genes in vitro (Gluzman et al., Cold Spring Harbor,
N.Y. 11724, p. 187), for the creation of transgenic animals
(WO95/22616), for the transfer of genes into cells ex vivo
(WO95/14785; WO95/06120) or for the transfer of genes into cells in
vivo (see in particular WO93/19191, WO94/24297, WO94/08026).
[0009] As regards the adeno-associated viruses (AAV), they are
relatively small DNA viruses which become integrated into the
genome of the cells which they infect, in a stable and relatively
site-specific manner. They are capable of infecting a broad
spectrum of cells, without inducing any effect on cell growth,
morphology or differentiation. Moreover, they do not seem to be
involved in pathologies in man. The genome of the AAVs has been
cloned, sequenced and characterized. It comprises about 4700 bases
and contains, at each end, an inverted terminal repeat (ITR) region
of about 145 bases which serves as replication origin for the
virus. The remainder of the genome is divided into 2 essential
regions carrying the encapsidation functions: the left part of the
genome, which contains the rep gene involved in the viral
replication and the expression of the viral genes; the right part
of the genome, which contains the cap gene encoding the virus
capsid proteins.
[0010] The use of vectors derived from AAVs for the transfer of
genes in vitro and in vivo has been described in the literature
(see in particular WO91/18088; WO93/09239; U.S. Pat. No. 4,797,368,
5,139,941, EP 488 528).
[0011] As regards the retroviruses, they are integrative viruses
which selectively infect dividing cells. They therefore constitute
vectors of interest for cancer or restenosis applications for
example. The genome of retroviruses essentially comprises two LTRs,
an encapsidation sequence and three coding regions (gag, pol and
env). The construction of recombinant vectors and their use in
vitro or in vivo has been widely described in the literature: see
in particular Breakfield et al., New Biologist 3 (1991) 203; EP
453242, EP 178220, Bernstein et al. Genet. Eng. 7 (1985) 235;
McCormick, BioTechnology 3 (1985) 689, and the like.
[0012] For their use as recombinant vectors, various constructs
derived from viruses have been prepared, incorporating various
genes of interest. In each of these constructs, the viral genome
was modified so as to make the virus incapable of autonomously
replicating in the infected cell. Thus, the constructs described in
the prior art are viruses which are defective for certain regions
of their genome which are essential for replication. In particular,
as regards adenoviruses, the first-generation constructs exhibit a
deletion in/of the E1 region, which is essential for viral
replication, at the level of which the heterologous DNA sequences
are inserted (Levrero et al., Gene 101 (1991) 195; Gosh-Choudhury
et al., Gene 50 (1986) 161). Moreover, to enhance the properties of
the vector, it has been proposed to create other deletions or
modifications in the adenovirus genome. Thus, a heat-sensitive
point mutation was introduced into the ts125 mutant, making it
possible to inactivate the 72 kDa DNA binding protein (DBP) encoded
by the E2 region (Van der Vliet et al., 1975). Other vectors
comprise a deletion of another region essential for the viral
replication and/or propagation, the E4 region. Adenoviral vectors
in which the E1 and E4 regions are deleted have highly reduced
transcription background noise and viral gene expression. Such
vectors have been described, for example, in applications
WO94/28152, WO95/02697, WO96/22378. Moreover, vectors carrying a
modification at the level of the IVa2 gene have also been described
(WO96/10088). In addition, so-called "minimum adenovirus" or
"pseudo-adenovirus" vectors (or alternatively Ad.DELTA.) containing
only the regions necessary in cis for the production of the virus
(ITR and encapsidation sequences) and lacking any coding viral
sequence have also been described (WO94/12649, WO94/28152,
WO95/02697), although their production remains very difficult, as
explained below.
[0013] As regards the AAVs, the vectors described generally lack
the entire coding regions Rep and Cap, which are replaced by
nucleic acids of interest.
[0014] In the recombinant vectors derived from retroviruses, the
gag, pol and env genes are generally deleted, completely or in
part, and replaced by a heterologous nucleic acid sequence of
interest. Moreover, the recombinant retroviruses may comprise
modifications at the level of the LTRs in order to suppress the
transcriptional activity, as well as large encapsidation sequences,
comprising part of the gag gene (Bender et al., J. Virol. 61 (1987)
1639).
[0015] Given their defective character in relation to the
replication, the production of these various recombinant viruses
involves the possibility of transcomplementing the functions
deleted from the genome. The transcomplementation is precisely the
source of major difficulties for the production of these viruses,
and in particular the provision of the transcomplementation
functions.
[0016] Two approaches have been developed in this regard. The first
is based on the construction of transcomplementing lines, that is
to say encapsidation lines. The second is based on the use of
helper adenoviruses or of helper plasmids.
[0017] Various encapsidation lines of defective viruses have been
constructed. These lines are capable of producing the functions
deficient in the viral vector. Generally, these lines comprise,
integrated into their genome, the region(s) deleted from the viral
genome (E1, E2 and/or E4 for example for the adenovirus; gag, pol
and/or env for the retrovirus, rep and/or cap for the AAV).
[0018] One of the lines known for the production of defective
adenoviruses is for example the line 293 into which part of the
adenovirus genome has-been integrated. More precisely, the line 293
is a human embryonic kidney cell line containing the left end
(about 11-12%) of the serotype 5 adenovirus (Ad5) genome,
comprising the left ITR, the encapsidation region, the E1 region,
including E1a and E1b, the region encoding the protein pIX and part
of the region encoding the protein pIVa2. This line is capable of
transcomplementing recombinant adenoviruses defective for the E1
region, that is to say lacking all or part of the E1 region, and of
producing viral stocks having high titres. This line is also
capable of producing, at permissive temperature (32.degree. C.),
virus stocks comprising, in addition, the heat-sensitive E2
mutation. Other cell lines capable of complementing the E1 region
have been described, based in particular on human lung carcinoma
cells A549 (WO94/28152) or on human retinoblasts (Hum. Gen. Ther.
(1996) 215). Moreover, lines capable of transcomplementing several
functions in the adenovirus have also been described. In
particular, there may be mentioned lines complementing the E1 and
E4 regions (Yeh et al., J. Virol. 70 (1996) 559; Cancer Gen. Ther.
2 (1995) 322; Krougliak et al., Hum. Gen. Ther. 6 (1995) 1575) and
lines complementing the E1 and E2 regions (WO94/28152, WO95/02697,
WO95/27071).
[0019] Various lines have also been described for the production of
defective retroviruses, generally capable of expressing the gag,
pol and env genes. Such lines are, for example, the PA317 line
(U.S. Pat. No. 4,861,719), the PsiCRIP line (WO90/02806), the
GP+envAm-12 line (WO89/07150), the BOSC line (WO94/19478) and the
like. To construct recombinant retroviruses comprising a nucleic
acid of interest, a plasmid comprising in particular the LTRs, the
encapsidation sequence and the said nucleic acid is constructed,
and then used to transfect an encapsidation line as described
above, capable of providing in trans the retroviral functions
deficient in the plasmid. The recombinant retroviruses produced are
then purified by conventional techniques.
[0020] The use of lines can however have certain disadvantages.
Thus, it is difficult, expensive and restrictive at the industrial
level to construct and to validate such lines. Indeed, these lines
should be stable and compatible with industrial uses. Furthermore,
the lines described hardly make it possible to avoid the production
of replicative contaminant viruses (RCA). Moreover, these lines do
not allow at the present time, in a satisfactory manner for an
industrial use, very highly defective viral genomes, such as for
example minimum adenoviruses as described above, to be
transcomplemented. Indeed, the adenovirus has a genome organized
into various transcription units whose spatiotemporal regulation is
very complex. It has so far not been possible to carry out
satisfactorily the transcomplementation of an adenovirus deleted of
all the coding viral sequences by expressing each transcription
unit separately, in a constitutive or conditional manner, using a
cell line. Thus, only a small proportion of the genome
corresponding to the E1, E4 and pIX regions, and to the three
proteins encoded by E2 (pol, DBP and p-TP) has been constitutively
expressed using cell lines. The remainder of the genome corresponds
to the major late transcription unit (MLTU) which produces all the
messengers for the structural and nonstructural proteins from a
primary transcript of 28 kb and is activated after replication of
the genome. Now, to generate minimum adenoviruses,
transcomplementation of these regions is essential. Neither do
these lines make it possible to obtain very high recombinant
retrovirus titres.
[0021] The second approach consists in cotransfecting with the
defective viral genome a construct (plasmid or adenovirus),
providing the complementation functions. In particular, the
defective recombinant AAVs are generally prepared by
cotransfection, in a cell line infected by a human helper virus
(for example an adenovirus), of a plasmid containing a nucleic
sequence of interest bordered by two AAV inverted terminal repeat
(ITR) regions, and of a plasmid carrying the AAV complementation
functions (rep and cap genes). Variants have been described in
applications WO95/14771; WO95/13365; WO95/13392 or WO95/06743. The
disadvantage of using a helper adenovirus lies mainly in the
increased risks of recombination between the adenoviral vector and
the helper adenovirus, and in the difficulty of separating the
recombinant from the helper during the production and purification
of the viral stocks. The disadvantage of using a helper plasmid,
for example a plasmid Rep/cap, lies in the transfection levels
obtained, which do not make it possible to produce high virus
titres.
[0022] The present application now describes a new system for the
production of viruses which makes it possible to overcome these
disadvantages. The system of the invention is based on the use of a
baculovirus to provide the complementation functions.
[0023] The production system according to the invention makes it
possible, in a particularly advantageous manner, to dispense with
the use of established complementation lines, to avoid the problems
of RCA, and to transcomplement highly defective genomes. In
addition, the system of the invention is applicable to any cell
capable of being infected by the desired virus and by a
baculovirus, and thus offers great flexibility of use.
[0024] A first subject of the invention therefore consists in a
process for the production of defective recombinant viruses
according to which the genome of the defective recombinant virus
and a baculovirus comprising all or some of the functions necessary
for the transcomplementation of the defective recombinant genome
are introduced into a population of competent cells.
[0025] The process of the invention is therefore based on the use
of a baculovirus to provide the complementing functions. Various
approaches are possible. It is possible, first of all, to use
competent cells not expressing any function of transcomplementation
of the defective recombinant genome. In this case, it is possible
to use either a baculovirus comprising all the functions necessary
for the transcomplementation of the defective recombinant genome,
or several baculoviruses each carrying one or more of the functions
necessary for the transcomplementation of the defective recombinant
genome. It is also possible to use a population of competent cells
capable of already transcomplementing one or more functions of the
defective recombinant genome (encapsidation line). In this case,
the baculovirus(es) used will provide only the functions necessary
for the transcomplementation of the defective recombinant genome
which are not already transcomplemented by the competent cells.
[0026] As indicated above, the advantages of the system of the
invention are numerous in terms of industrialization (no need for
lines, no RCA, and the like), and in terms of applications
(production of recombinant viruses carrying any type of deletion,
and particularly of highly defective recombinant adenoviruses). In
addition, since the baculovirus does not replicate in human cells,
the viral preparation obtained is not contaminated by the
baculovirus. Furthermore, the baculovirus being phylogenetically
very distant from adenoviruses, there is no risk of recombination
or transcomplementation between the two. This system therefore
makes it possible, in an advantageous manner, to produce
concentrated stocks of defective viruses, lacking RCA. This system
is most particularly advantageous for the production of defective
recombinant adenoviruses.
[0027] Baculoviruses are enveloped, circular double-stranded DNA
viruses specific for invertebrates. Their prototype, AcNPV, has a
genome of 133 kb. It is widely used as vector for the expression of
eukaryotic genes in insect cells, starting from two strong
promoters [polyhedrin (Ph) and P10], (King and Possee, The
baculovirus expression system. London: Chapman & Hall, 1992.)
AcNPV is capable of infecting some mammalian cells, but the genome
is neither transcribed nor translated. Recently, Hofmann et al.
(PNAS 92 (1995) 10099) have shown that in vitro, hepatocytic cells
can be transduced by a purified recombinant baculovirus expressing
the LacZ gene. No cellular toxicity was reported, even with a
multiplicity of infection of 1000, and the transfection efficiency
described in this article is about 50% for an MOI of 100.
[0028] The applicant has now shown that it is possible to infect
various cell types with a recombinant baculovirus. In particular,
the applicant has shown that it was possible, with a recombinant
baculovirus, to infect cells of human origin such as immortalized
embryonic cells. The applicant has also shown that it is possible
to obtain a very high transduction efficiency (>80%). The
applicant has also shown that it is possible to introduce, into a
baculovirus, functions for complementation of an adenovirus, and to
express these functions in a population of competent cells. The
applicant has thus made it possible to show that the baculovirus
constitutes an inert vector which can be advantageously used for
the transfer and expression of virus complementation functions into
mammalian, particularly human, cells. Other advantages of the
system of the invention are in particular (i) the large cloning
capacity which makes it possible to complement a whole adenoviral
genome and (ii) the advanced development of the technology of the
baculovirus.
[0029] The baculovirus carrying the functions for complementation
of the virus is also designated in the text which follows helper
baculovirus. It may comprise various functions for complementation
of the virus.
[0030] Thus, the helper baculovirus may comprise the E1 region of
the adenovirus. A Baculo-E1 can be used for the production of
first-generation adenoviruses, that is to say adenoviruses
defective for the E1 region (Ad.DELTA.E1), regardless of its E3
status (i.e. defective Ad.DELTA.E1, .DELTA.E3, or not). The
production of first-generation defective recombinant adenoviruses
(defective for the E1, and possibly E3, region) constitutes a first
particularly advantageous application of the process of the
invention. As indicated above, various lines have been described in
the literature which are capable of transcomplementing the E1
function (cells 293, cells A549, cells 911, and the like). However,
various zones of homology exist between the region carrying the
transcomplementation functions which is integrated into the genome
of the line and the DNA of the recombinant virus which it is
desired to produce. Because of this, during production, various
recombination events may occur, generating replicative viral
particles, in particular type E1+adenoviruses. This may be a single
recombination event followed by breaking of the chromosome, or a
double recombination. These two types of modification lead to
reintegrating into its initial locus within the adenoviral genome
the E1 region contained in the cellular genome. Moreover, given the
high titres of recombinant vector which are produced by the line
293 (greater than 10.sup.12), the probability of these
recombination events occurring is high. In fact, it has been
observed that numerous batches of first-generation defective
recombinant adenoviral vectors were contaminated by replicative
viral particles, which may constitute a major disadvantage for
pharmaceutical uses. Indeed, the presence of such particles in
therapeutic compositions would induce in vivo an uncontrolled viral
propagation and dissemination with risks of inflammatory reaction,
of recombination and the like. The contaminated batches cannot
therefore be used in human therapy.
[0031] The present invention makes it possible to overcome these
disadvantages. Indeed, according to one embodiment of the process
of the invention, the genome of the recombinant adenovirus
defective for the E1, and possibly E3, region is introduced into
the competent cells, these cells are infected, simultaneously or
otherwise, with a baculovirus comprising the E1 region, the
adenovirus E1 region present in the baculovirus and the genome of
the defective recombinant adenovirus comprising no zone of homology
(overlapping) capable of giving rise to recombination. According to
this embodiment, it is thus possible to rapidly produce, without an
established line, stocks of first-generation recombinant
adenoviruses free of RCA. Moreover, as indicated below, the stocks
of recombinant adenoviruses thus generated, free of RCA, can be
used as starting material for a new production, by coinfection in
the competent cells with a baculovirus.
[0032] The helper baculovirus may also comprise the E2 region of
the adenovirus, in full or in part, particularly the E2a and/or E2b
region. A baculo-E2 may be used to produce, in competent cells,
adenoviruses defective for the E2 region (Ad-.DELTA.E2), and
possibly for the E3 region (Ad-.DELTA.E2, .DELTA.E3). In addition,
in competent cells capable of complementing the E1 region of the
adenovirus, the baculo-E2 may allow the production of recombinant
adenoviruses defective for the E1 and E2 (Ad-.DELTA.E1, .DELTA.E2)
and possibly E3 (Ad-.DELTA.E1, .DELTA.E2, .DELTA.E3) regions.
Likewise, in competent cells capable of complementing the E1 and E4
regions of the adenovirus (for example in IGRP2 cells), the
baculo-E2 may allow the production of recombinant adenoviruses
defective for the E1, E2 and E4 (Ad-.DELTA.E1, .DELTA.E2,
.DELTA.E4) and possibly E3 (Ad-.DELTA.E1, .DELTA.E2, .DELTA.E3,
.DELTA.E4) regions.
[0033] The helper baculovirus may also comprise the E4 region (in
full or in part) of the adenovirus. A baculo-E4 may be used to
produce, in competent cells, adenoviruses defective for the E4
region (Ad-.DELTA.E4), and possibly for the E3 region
(Ad-.DELTA.E4, .DELTA.E3). In addition, in competent cells capable
of complementing the E1 region of the adenovirus, the baculo-E4 may
allow the production of recombinant adenoviruses defective for the
E1 and E4 (Ad-.DELTA.E1, .DELTA.E4) and possibly E3 (Ad-.DELTA.E1,
.DELTA.E4, .DELTA.E3) regions.
[0034] The helper baculovirus may also comprise the E1 and E4
regions (in full or in part) of the adenovirus. A baculo-E1, E4 may
be used to produce, in competent cells, adenoviruses defective for
the E1 and E4 (Ad-.DELTA.E1, .DELTA.E4) and possibly E3
(Ad-.DELTA.E1, .DELTA.E4, .DELTA.E3) regions, as illustrated in
FIG. 1.
[0035] In addition, to generate viruses defective for the E1 and E4
regions, it is also possible to use two helper baculoviruses, one
expressing the E1 function, the other the E4 function, in full or
in part.
[0036] In the same manner, the helper baculovirus may comprise the
E1, E2 and E4 regions (in full or in part), and possibly the
regions carrying the late genes (L1-L5).
[0037] The helper baculovirus may also comprise the AAV Rep and/or
Cap regions. A baculo-Rep/Cap thus makes it possible to complement,
in a line of competent cells, an AAV genome lacking any coding
viral sequence (FIG. 5).
[0038] The baculovirus may also comprise the gag, pol and/or env
regions of a retrovirus. A baculo-gag/pol/env thus makes it
possible to complement, in a line of competent cells, a retroviral
genome lacking any coding viral sequence.
[0039] It is also possible to use a baculovirus comprising the
gag/pol regions and a second baculovirus containing the env
region.
[0040] In general, it is preferable that the genome of the
defective recombinant virus and the complementation regions present
in the baculovirus do not overlap. This makes it possible, indeed,
to avoid the risks of recombination and thus the generation of RCA.
This is particularly important for the generation of
first-generation adenoviruses (Ad-.DELTA.E1). In this case, the E1
region introduced into the baculovirus is defined so that it does
not possess any common sequence with the recombinant genome. To do
this, it is possible, for example, to delete from the recombinant
genome a region larger than the complementing region inserted into
the baculovirus, as illustrated in the examples. This is also
advantageous for the generation of Ad-.DELTA.E1, .DELTA.E4
adenovirus.
[0041] Thus, in a specific embodiment of the process of the
invention, the genome of the defective recombinant virus is
introduced into the competent cells, these cells are infected,
simultaneously or otherwise, with a baculovirus comprising all or
some of the functions necessary for the complementation of the
defective genome, the complementation functions present in the
baculovirus and the genome of the defective recombinant virus
comprising no zone of homology capable of giving rise to
recombination. Advantageously, the viral genome is a recombinant
adenovirus genome defective for the E1 region and the baculovirus
carries a region of the adenovirus capable of transcomplementing
the E1 region. According to another variant, the viral genome is a
recombinant adenovirus genome defective for the E1 and E4 regions
and the baculovirus carries two adenovirus regions capable of
transcomplementing the said regions or two baculoviruses are used,
one carrying a region of the adenovirus capable of
transcomplementing the E1 region and the other a region of the
adenovirus capable of transcomplementing the E4 region, without
zones of homology with the defective adenoviral genome.
[0042] According to a specific embodiment, all the coding regions
of the adenovirus are carried by one or more helper baculoviruses.
According to a more specific embodiment, only one helper
baculovirus comprising all the coding regions of the adenovirus is
used. Such a helper baculovirus can thus be used to transcomplement
minimum recombinant adenoviruses. Such a baculovirus may in
particular comprise the whole of one adenoviral genome, with the
exception of the encapsidation region and possibly the ITRS, as
illustrated in the examples.
Preparation of the Complementation Functions
[0043] The complementation functions introduced into the helper
baculovirus may be derived from viruses of different serotypes.
[0044] As regards adenoviruses, various serotypes exist whose
structure and properties vary somewhat, but which exhibit a
comparable genetic organization. More particularly, the
complementation functions used for the construction of the
baculoviruses according to the invention are derived from an
adenovirus of human or animal origin.
[0045] As regards adenoviruses of human origin, there may be
mentioned, preferably, those classified in the C group. Still more
preferably, among the various human adenovirus serotypes, the use
of the type 2 or 5 adenoviruses (Ad2 or Ad5) is preferred within
the framework of the present invention. It is also possible to use
regions derived from type 7 or 12 adenoviruses, belonging to groups
A and B. Among the various adenoviruses of animal origin, the use
of the adenoviruses of canine origin, and particularly all the
strains of the CAV2 adenoviruses [Manhattan or A26/61 strain (ATCC
VR-800) for example] is preferred within the framework of the
invention. Other adenoviruses of animal origin are mentioned in
particular in application WO94/26914 incorporated into the present
by reference.
[0046] In a preferred embodiment of the invention, the
complementation function is derived from a group C human adenovirus
genome. More preferably, it is derived from the genome of an Ad2 or
Ad5 adenovirus.
[0047] The regions carrying the various complementation functions
may be obtained, from an adenoviral genome, by enzymatic cleavages
according to methods known to persons skilled in the art. These
regions may optionally be modified in order to reduce their size,
or to replace certain regulatory elements (promoter, enhancer and
the like) with heterologous elements. In general, these regions are
prepared as follows: the DNA of an adenovirus is purified by
caesium chloride gradient centrifugation or obtained in vitro from
a prokaryotic (WO96/25506) or eukaryotic (WO95/03400) plasmid. The
DNA is then cleaved with appropriate restriction enzymes and the
fragments obtained, carrying the desired complementation functions,
are identified and selected. The choice of the restriction enzymes
used depends on the desired complementation functions. It is then
guided by the restriction maps and the published sequences of the
adenoviral genomes. Thus, the E1 region may be isolated in the form
of fragments carrying all the reading frames of E1A and E1B
downstream of the E1A promoter. The E4 region may be isolated in
the form of fragments carrying the whole of the reading frames, or
only part of them, and preferably the frames ORF3 or ORF6 or
ORF6-ORF6/7.
[0048] Similar methodologies are used to prepare the AAV and
recombinant retrovirus complementation regions. Thus, the AAV rep
and/or cap regions may be obtained by enzymatic cleavage from the
viral DNA isolated from various AAV serotypes. This is preferably
AAV-2. For retroviruses, the gag, pol and/or env regions may also
be obtained according to conventional molecular biological
techniques, from various types of retroviruses, such as in
particular MoMuLV (Murine Moloney Leukaemia Virus; also called
MoMLV), MSV (Murine Moloney Sarcoma Virus), HaSV (Harvey Sarcoma
virus); SNV (Spleen Necrosis Virus), RSV (Rous Sarcoma Virus) or
Friend's virus.
Construction of the Helper Baculovirus
[0049] The fragments carrying the complementation regions are then
subcloned into a plasmid vector allowing their manipulation (finer
digestions, PCR, additions of regulatory sequences, and the like),
for example in a prokaryotic or eukaryotic organism. The final
fragment obtained, encoding the complementation function(s) is then
introduced into the helper baculovirus using conventional molecular
biological techniques. Specifically, the fragment is cloned between
two sequences homologous to a region of the genome of a
baculovirus, and then the resulting fragment or plasmid is
cotransfected with the genome of a baculovirus into insect cells
(conventionally Sf9 and Sf21, spodoptera frugiperda cells, but also
Tn-368 and High-Five.TM. BTI-TN-5B1-4 (Gibco), trichopulsia ni
cells, or any other insect cell permissive to baculoviruses and
capable of being used for their production). The homologous
recombination between the plasmid or fragment and the genome of the
baculovirus generates the desired recombinant baculovirus, which
may be recovered and purified according to conventional methods
(see in particular King and Possee: the baculovirus expression
system. London: Chapman & Hall, 1992). For the construction of
the recombinant baculoviruses, various kits comprising shuttle
vectors are commercialized and may be used according to the
recommendations of the manufacturers. In particular, it is possible
to use the shuttle vectors pBAC marketed by the company Clontech,
the vectors pAc (Verne et al., Bio/Technology 6 (1988) 47,
Pharmingen, USA), the vectors pBlue-Bac (Invitrogen) or the vectors
pBSV (Boehringer). The complementation functions may thus be
inserted into different sites of the baculovirus, and in particular
into the locus of the polyhedrin gene or of the p10 gene. Moreover,
various baculovirus strains can be used, such as in particular
AcNPV or Bombyx mori (Maeda et al., Nature 315 (1988) 592).
Furthermore, the baculovirus used may be modified to enhance/change
its tropism. It is indeed possible to modulate the tropism of the
viral vectors by modifying their surface proteins so as (i) to
limit it by fusion of the viral proteins with a specific ligand
(light immunoglobulin chain, Gastrin-Releasing Peptide) or (ii) to
broaden it by formation of pseudotypes with a heterologous viral
glycoprotein [G of the Vesicular Stomatitis Virus (VSV)], [Liu et
al., J. Virol 70(4) (1996) 2497; Michael et al., Gene Ther. 2
(1995) 660]. Recently, it was shown that the baculovirus surface
glycoprotein (gp64) fused with gp120 of the HIV virus was capable
of binding to the CD4 receptor (Boublik et al. Bio/Technology 13
(1995) 1079). This modification of gp64 does not affect the
viability of the baculovirus in insect cells. A similar construct
with the G of VSV should make it possible to enhance the tropism of
the baculovirus for mammalian cells and therefore to increase the
transduction efficiency of the Huh7 cells as well as other cell
lines.
[0050] In the helper baculovirus, the complementation functions are
advantageously placed under the control of a heterologous promoter
(i.e. of a different origin from the baculovirus), which is
functional in competent cells. It appears, indeed, that the
baculovirus promoters do not make it possible to obtain sufficient
levels of expression of the complementation functions in cells
other than insect cells, and are therefore not the most appropriate
for the applications of the invention. The promoter may first of
all be the actual promoter (homologous promoter) of the
complementation functions of the virus (E1A, E4, E2, MLP promoter
for the adenovirus, P5 or P19 promoters of AAV, the LTR promoter of
RSV, and the like). It may also be any promoter of different origin
which is functional in the competent cell used. To this effect,
there may be mentioned for example the promoters of genes expressed
in this cell, or known ubiquitous promoters such as for example the
promoter of the PGK gene, the immediate-early promoter of CMV, the
promoter of the TK gene of the herpesvirus or alternatively the LTR
promoter of RSV. It may also be a regulated promoter, such as for
example the promoter of the MMTV virus, a promoter responding to
hormones, for example of the GRE5 type, or a promoter regulated by
tetracycline (WO). Advantageously, it is an inducible or strong
ubiquitous, homologous promoter.
[0051] Thus, another subject of the present invention relates to a
recombinant baculovirus comprising, inserted into its genome, a
nucleic acid encoding a complementation function of a virus placed
under the control of a heterologous promoter. More particularly,
the complementation function is a protein necessary for the
production of the said virus, and whose coding region is inactive
(mutated, deleted and the like) in the defective viral genome. For
adenoviruses, the complementation function is more particularly
chosen from all or some of the functions encoded by the E1, E2, E4,
L1-L5, pIX and IVa2 regions of the adenovirus, alone or in
combination. For the AAV, they are functions encoded by the Rep
and/or Cap regions; and for the retrovirus, gag, pol and/or env.
Advantageously, the nucleic acid corresponds to a region of a viral
genome comprising the region encoding the complementation function
chosen. In particular, it is a fragment of a genome of adenoviruses
of serotype Ad2 or Ad5, MOMLV or AAV-2. In a particularly preferred
manner, the nucleic acid also comprises the promoter region which
is naturally responsible for the expression of the complementation
functions chosen.
[0052] By way of a specific example, the present invention relates
to a baculovirus comprising all or part of the E1 region of an
adenovirus. More particularly, it is a baculovirus comprising the
E1a, E1b or E1a and E1b region. The E1 region of the adenovirus is
located at the level of nucleotides 104 (promoter E1a) to 4070
(polyA E1b) of Ad5. In particular, the TATA box of the E1a promoter
is located at the level of nucleotide 470, the ATG codon of E1a at
the level of nucleotide 560, and the stop codon E1b at the level of
nucleotide 3511. There may be mentioned by way of precise example a
baculovirus comprising a fragment 391-3511. This helper baculovirus
is particularly suitable for the production of recombinant
adenoviruses defective for the E1 region, carrying a larger
deletion than this 391-3511 fragment. In particular, it is suitable
for the production of first-generation adenoviruses, without RCA,
carrying a deletion in the E1 region covering nucleotides 383-3512
inclusive.
[0053] Another specific example of a baculovirus according to the
invention comprises, for example, all or some of the E1 and E4
regions of the adenovirus. The E4 region of the adenovirus consists
of 7 open reading frames, designated ORF1, ORF2, ORF3, ORF4,
ORF3/4, ORF6 and ORF6/7. Among the proteins encoded by these
various ORFs, those produced by ORF3 and ORF6 appear to allow the
"replication" of the virus, and therefore the transcomplementation
of an adenovirus defective for the E4 region, even in its entirety.
As a result, the helper baculovirus of the invention advantageously
comprises all the E4 region or only part thereof comprising at
least the ORF3 or ORF6 frame. The various parts of the E4 region
may be obtained by enzymatic cleavages or modified according to
methods known to persons skilled in the art. In particular, the
reading frame ORF6 may be isolated from the E4 region in the form
of a BglII-PvuII fragment, corresponding to nucleotides
34115-33126, and the reading frames ORF6-ORF6/7 may be isolated
from the E4 region in the form of a BglII-BglII fragment
corresponding to nucleotides 34115-32490 of the genome of Ad5. The
baculovirus may also comprise the whole of the reading frames
ORF1-ORF7 (for example in the form of a 32800-35826 or 32811-35614
or 32811-35640 fragment). It is understood that other fragments may
be determined on the basis of published sequences of the adenoviral
genomes. The use of a baculovirus carrying a reduced unit of the E4
region is advantageous because it allows the transcomplementation
of a defective adenoviral genome carrying a larger deletion of the
E4 region, therefore without a zone of homology, and thus to avoid
any possibility of recombination.
[0054] According to a first embodiment, the nucleic acid encoding
the complementation function(s) is introduced into the helper
baculovirus in the form of an expression cassette. This embodiment
is the easiest to use. It is particularly suitable for the
production of recombinant adenoviruses defective for
immediate-early genes and for the production of defective
recombinant AAVs and retroviruses.
[0055] According to another embodiment, the nucleic acid encoding
the complementation function(s) is introduced into the helper
baculovirus in the form of an excisable cassette, generating a
replicative molecule in the competent cell. The replication of the
cassette in the cell makes it possible advantageously to increase
the copy number of the complementing genes, and thus to enhance the
production levels of the system. This embodiment is particularly
suitable for the production of very highly defective recombinant
adenoviruses, particularly defective for the structural genes. In
particular, this embodiment is particularly suitable for the
production of "minimum" adenoviruses. Indeed, the quantity of
structural protein is a limiting factor for the production of high
titres of highly defective adenoviruses (minimum adenovirus type).
This embodiment makes it possible, for the first time, to
considerably increase the intracellular levels of
transcomplementing proteins, particularly of structural proteins of
the adenovirus (encoded by the L1 to L5 regions), up to levels
compatible with the transcomplementation of minimum
adenoviruses.
[0056] Thus, the applicant has shown that it is possible to
construct recombinant baculoviruses comprising a heterologous
region capable of being excised in a cell, preferably in an
inducible and regulated manner, in order to generate a circular and
replicative molecule (of episomal type).
[0057] The excision is advantageously carried out by a
site-specific recombination mechanism, and the replication in the
cell is brought about by a replication origin, independent of the
state of cell division.
[0058] More preferably, the site-specific recombination used
according to the process of the invention is obtained by means of
two specific sequences which are capable of recombining with each
other in the presence of a specific protein, generally called
recombinase. These specific sequences, arranged in the appropriate
orientation, flank in the baculovirus the sequences encoding the
complementation functions. Thus, the subject of the invention is
also a recombinant baculovirus comprising, inserted into its
genome, at least one DNA region flanked by two sequences allowing a
site-specific recombination and positioned in direct orientation,
the said DNA region comprising at least one replication origin
functional in competent cells and a nucleic acid encoding a
complementation function of a virus.
[0059] The sequences allowing the recombination which are used in
the framework of the invention generally comprise from 5 to 100
base pairs, and more preferably less than 50 base pairs. They may
belong to different structural classes, and in particular to the
family of the recombinase of the P1 bacteriophage or of the
resolvase of a transposon.
[0060] Among the recombinases belonging to the bacteriophage 1
integrase family, there may be mentioned in particular the
integrase of phages lambda (Landy et al., Science 197 (1977) 1147),
P22 and F80 (Leong et al., J. Biol. Chem. 260 (1985) 4468), HP1 of
Haemophilus influenzae (Hauser et al., J. Biol. Chem. 267 (1992)
6859), the Cre integrase of the P1 phage, the integrase of the
plasmid pSAM2 (EP 350 341) or the FLP recombinase of the plasmid 2
.mu.m of the yeast Saccharomyces cerevisiae.
[0061] Among the recombinases belonging to the Tn3 transposon
family, there may be mentioned in particular the resolvase of the
Tn3 transposon or of the gd, Tn21 and Tn522 transposons (Stark et
al., 1992); the Gin invertase of the mu bacteriophage or the
resolvase of plasmids, such as that of the fragment par of RP4
(Abert et al., Mol. Microbiol. 12 (1994) 131).
[0062] According to a preferred embodiment, in the recombinant
baculoviruses of the present invention, the sequences allowing the
site-specific recombination are derived from a bacteriophage. More
preferably, they are sequences for attachment (attP and attB
sequences) of a bacteriophage or of derived sequences. These
sequences are capable of specifically recombining with each other
in the presence of a recombinase called integrase. By way of
specific examples, there may be mentioned in particular the
sequences for attachment of the phages lambda, P22, F80, P1, HP1 of
Haemophilus influenzae or of the plasmid pSAM2, or 2 .mu.m.
[0063] Still more preferably, the sequences allowing the
site-specific recombination are represented by the recombination
system of the P1 phage. The P1 phage possesses a recombinase called
Cre which specifically recognizes a nucleotide sequence of 34 base
pairs called lox P site (Sternberg et al., J. Mol. Biol. 150 (1981)
467). This sequence is composed of two palindromic sequences of 13
bp separated by a conserved sequence of 8 bp. The site-specific
recombination is advantageously obtained using LoxP sequences or
derived sequences, and the Cre recombinanse.
[0064] The term derived sequence includes the sequences obtained by
modification(s) of the recombination sequences above, conserving
the capacity to specifically recombine in the presence of the
appropriate recombinase. Thus, it may involve reduced fragments of
these sequences or on the contrary fragments extended by addition
of other sequences (restriction sites and the like). It may also
involve variants obtained by mutation(s), particularly by point
mutation(s).
[0065] According to a preferred embodiment of the invention, the
sequences allowing a site-specific recombination are therefore LoxP
sequences of the P1 bacteriophage, and the recombination is
obtained in the presence of the Cre protein. In this regard, the
recombination may be obtained by bringing the competent cells
directly into contact with the Cre recombinase, or by expression of
the gene encoding the Cre recombinase in the competent cells.
Advantageously, the Cre recombinase is produced in the cell by
inducing the expression of the corresponding gene. Thus, the gene
encoding the recombinase is advantageously placed under the control
of an inducible promoter, or constructed in a regulatable form. In
this regard, there is advantageously used a fusion between Cre and
the steroid hormone (oestradiol, progesterone and the like) binding
domain allowing the activity of Cre to be regulated and therefore
the recombination event to be induced (Metzger et al., PNAS 92
(1995) 6991). More generally, the expression of the recombinase may
be controlled by any strong promoter, regulated or otherwise. The
expression cassette may be transfected into the competent cells, or
integrated into the genome of the competent cells, as illustrated
in the examples.
[0066] This system therefore makes it possible to generate
replicative molecules producing, in the competent cells, high
levels of virus, particularly adenovirus, complementation function.
This type of construct is particularly suitable for the
complementation of highly defective genomes, in particular of
adenoviral genomes defective for the late genes. Thus, a specific
construct according to the invention is represented by a
baculovirus comprising, inserted into its genome, at least a DNA
region flanked by two LoxP sequences positioned in direct
orientation, the said DNA region comprising at least one
replication origin functional in the competent cells and one
nucleic acid encoding a complementation function of an adenovirus.
Advantageously, the complementation functions comprise all or some
of the immediate-early genes present in the E1, E2 and E4 regions.
Still more preferably, the complementation functions comprise all
or some of the immediate-early genes and of the delayed-early
genes. Preferably, the complementation functions allow the
complementation of a recombinant adenovirus lacking any coding
viral sequence. In particular, the complementation functions
consist of the whole of the adenoviral genome, with the exception
of the ITRs and of the packaging region. According to a specific
variant, the complementation functions consist of a complete
adenoviral genome lacking, however, the packaging region (Ad.Psi-).
This genome comprises in particular the ITRs which serve for the
replication of the genome in the competent cells, after
excision.
[0067] To bring about the replication of the episomal molecule, the
latter therefore contains a replication origin functional in the
competent cells used. This replication origin preferably consists
of the actual ITR sequences of the adenovirus, which allow
substantial amplification of the molecule. It may also be another
replication origin allowing, preferably, amplification by a factor
greater than 20 of the viral DNA in the competent cell. There may
be mentioned, by way of illustration, the origin OriP/EBNA1 of the
EBV virus or the E2 region of the papilloma virus. It is understood
that the ITR sequences of the adenovirus constitute a preferred
embodiment.
[0068] For carrying out the process of the invention, the helper
baculovirus(es) are generally used at a Multiplicity of Infection
(MOI) allowing a large population of cells to be infected, without
significantly impairing cell viability. Generally, it is more
particularly between 10 and 1000. The MOI corresponds to the number
of viral particles per cell. The MOI may be easily adjusted by
persons skilled in the art depending on the competent cells used,
essentially on the basis of two criteria: the infection efficiency
and the possible toxicity. Advantageously, the MOI used for the
helper baculovirus is between 20 and 500.
Introduction of the Viral Genome
[0069] As indicated above, the process of the invention comprises
the introduction, into competent cells, of the helper baculovirus
and of the recombinant viral genome. In this regard, the genome of
the defective recombinant adenovirus may be introduced in various
ways into the competent cell.
[0070] It may, first of all, be a purified defective recombinant
adenovirus, advantageously free of RCA. In this case, the competent
cells are infected with the defective recombinant adenovirus and
with the helper baculovirus. The infection with the recombinant
adenovirus makes it possible to introduce into the competent cell
the corresponding genome, which is then amplified and encapsidated
in order to produce stocks at a high titre, free of RCA. This
embodiment is particularly advantageous for generating
first-generation viruses (Ad-.DELTA.E1; Ad-.DELTA.E1, .DELTA.E3).
Indeed, these viruses are difficult to produce at high titres,
without contamination with RCAs. According to the process of the
invention, it is now possible, starting with a first-generation
defective recombinant adenovirus, by coinfection in a competent
cell with a baculovirus comprising the E1 region, to obtain
concentrated stocks, of high quality. This embodiment is also
advantageous for the production of viruses defective in two or
three essential regions of their genome (E1, E2, E4 in particular).
In general, this embodiment is advantageous because the efficiency
of infection with the adenovirus is very high (greater than the
efficiency of transfection with DNA), and therefore makes it
possible to generate concentrated stocks. In this embodiment, the
recombinant adenovirus and the recombinant baculoviruses are used
at multiplicities of infection (MOI) allowing a large population of
cells to be infected, without significantly impairing cell
viability. The MOI used for the baculovirus is that stated above
(between 10 and 1000). As regards the adenoviruses, it is
advantageously between 1 and 1000, preferably between 1 and 500,
still more preferably between 1 and 100. The MOI used for the
adenovirus is also adjusted according to the cell type chosen. The
MOI range may be easily determined by persons skilled in the art
using, for example, an adenovirus and a baculovirus comprising a
separate marker gene, in order to measure the efficiency of
infection and any competition. More preferably, the MOI of the
adenovirus is less than 50, for example between 1 and 20.
[0071] According to another particularly advantageous embodiment,
the genome of the defective recombinant adenovirus is introduced in
the form of DNA. In this case, the genome is introduced by
transfection, optionally in the presence of a
transfection-facilitating agent (lipids, calcium phosphate and the
like). The recombinant genome thus introduced may be prepared in
vitro according to various techniques, and in particular in E. coli
(WO96/25506) or in a yeast (WO95/03400). This embodiment is in
particular useful for generating a first batch of defective
recombinant virus, free of RCA, which can then in turn be used to
produce stocks with a high titre according to the preceding
embodiment.
[0072] The genome of the defective recombinant adenovirus may also
be introduced using another recombinant baculovirus. According to
this embodiment, the genome of the defective recombinant adenovirus
is prepared in vitro, for example as indicated above, and then
introduced into a baculovirus, in the form of a cassette capable of
being excised in the competent cell. According to this embodiment,
the competent cells are put in the presence of a baculovirus
carrying the genome of the defective recombinant adenovirus, and of
one or more helper baculoviruses (carrying the complementation
functions). This embodiment is particularly advantageous for the
production of highly defective recombinant adenoviruses. By virtue
of this system, it is indeed possible to introduce into the
population of competent cells high quantities both of the highly
defective recombinant genome and of the corresponding
complementation functions.
[0073] In this regard, a process of the invention therefore
comprises the coinfection of competent cells with a baculovirus
carrying the genome of the defective recombinant adenovirus, and
one or more helper baculoviruses carrying the complementation
functions. The MOI values used in this embodiment are also between
10 and 1000 for each of the baculoviruses used.
[0074] Two types of constructs have been prepared in the prior art
for the production of minimum adenoviruses: (1) the transgene
(.beta.-galactosidase) cloned between the ITRs, bordered by a
unique restriction site or (2) the right and left ITRs cloned in
direct orientation in 5' of the transgene (Fisher et al., Virology
217 (1996) 11; Kumar-Singh et al., Hum. Mol. Genet. 5 (1996) 913).
Minimum adenoviruses were produced in the cells 293 by transfection
of linearized (1) or circular (2) DNA, the viral proteins necessary
for the replication and for the encapsidation of the minigenome
being provided in trans by a helper virus (Ad.DELTA.E1). The
minimum adenoviruses behave like interfering defective (ID)
particles and are progressively amplified during successive
passages. The major problem posed by the use of this methodology is
the separation of the two types of particles produced, responsible
for the contamination of the stocks by the helper virus, and the
very low titres of minimum adenoviruses thus obtained (less than
10.sup.8 pfu/ml).
[0075] The present application makes it possible, for the first
time, to generate minimum adenoviruses using a baculovirus to
deliver the adenoviral minigenome and a baculovirus to provide all
the transcomplementation functions (complementing genome).
[0076] The recombinant adenoviral genome is advantageously
introduced into the baculovirus, between two sequences allowing a
site-specific recombination in the competent cells, as described
for the helper baculovirus.
[0077] The present application describes in particular a system for
the production of a minimum adenovirus using a baculovirus to
deliver the adenoviral minigenome with the aid of the loxP/Cre
system and a baculovirus to provide all the transcomplementation
functions (complementing genome), also with the aid of a Cre/loxP
system (see FIGS. 2-4).
[0078] According to another embodiment, the site-specific
recombination system used to deliver the complementation functions
is different from that used to deliver the genome of the
recombinant adenovirus. In particular, the LoxP/Cre system may be
used to deliver the defective adenoviral genome and the AttP/AttB
system to deliver the complementation function(s).
[0079] The process of the invention thus makes it possible to
construct an adenoviral vector deleted of all coding viral
sequences and comprising only the ITRs and the encapsidation signal
(minimum adenoviruses). This vector can theoretically accommodate
up to 37 kb of exogenous sequence whereas the cloning capacity of
current vectors does not exceed 8.5 kb. It thus makes it possible
to clone genes of large size such as the dystrophin gene (14 kb)
with all their regulatory elements (promoter, enhancer, introns and
the like) so as to obtain an optimum expression, in the target
tissue. Furthermore, the absence of any immunogenic viral sequence
should increase the duration of expression of the transgene in
quiescent tissues.
[0080] The genome of AAV or the defective retrovirus may also be
introduced in the form of a virus, a genome or a plasmid, according
to the techniques described above.
Competent Cells
[0081] The process of the invention may be carried out in various
types of cells. For the purposes of the invention, "competent cell"
is understood to mean a cell permissive to infection by the
baculovirus and the virus to be produced, and allowing a productive
viral cycle for the latter. The capacity to infect cells with these
viruses can be determined using recombinant viruses expressing a
marker gene such as the E. coli LacZ gene. It is preferably a
mammalian cell, still more preferably a cell of human origin. The
competent cells used may be quiescent cells or actively dividing
cells, established lines or primary cultures. They are
advantageously mammalian cells compatible with an industrial use,
that is to say without a known pathogenic character, capable of
being cultured and, where appropriate, of being stored under
appropriate conditions. Advantageously, the cells used are hepatic,
muscular, fibroblastic, embryonic, nerve, epithelial (pulmonary) or
ocular (retinal) cells. There may be mentioned, by way of
nonlimiting example, the cells 293 or any derived cell comprising
an additional complementation function (293E4, 293E2a, and the
like), the A549 cells, the HuH7 cells, the Hep3B cells, the HepG2
cells, the human retinoblastic cells (HER, 911), the HeLa cells,
the 3T3 cells or the KB cells.
[0082] To carry out the process of the invention, the genome of the
recombinant virus and the baculovirus may be introduced into the
population of competent cells simultaneously or spaced out over
time. Advantageously, the cells are brought into contact both with
the recombinant genome and the helper baculovirus. In the case of a
system generating replicative molecules in vivo, the recombinase is
introduced or expressed beforehand, simultaneously or
subsequently.
[0083] The production of the viruses generally leads to the lysis
of the cells. The viruses produced can therefore be harvested after
cell lysis, according to known purification methods. They can then
be packaged in various ways depending on the desired use. Moreover,
to avoid any risk of contamination of the viral stock with possible
traces of baculoviruses that have not penetrated into the competent
cells (helper baculovirus or baculovirus providing the recombinant
viral genome), it is possible to apply the following
techniques:
[0084] it is possible to purify the adenoviruses by chromatography
according to the method described in application FR96/08164. This
technique makes it possible to separate the adenovirus from any
possible residual baculovirus;
[0085] it is also possible to cause organic solvents (for example
ether, chloroform) to act on the stocks of purified adenovirus.
Indeed, the baculovirus is an enveloped virus (glycoprotein
envelope), and is therefore very sensitive to any organic solvent
(which extracts the lipids from its envelope); in contrast, the
adenovirus is not enveloped, and the same solvents have no effect
on it;
[0086] it is also possible, by CsCl gradient purification, to
separate, by density, any residual baculovirus and the recombinant
virus.
[0087] These three methods can be used independently or conjointly.
Moreover, any other method known to a person skilled in the art can
also be used.
Use of the Viruses
[0088] The viruses thus produced can be used for the cloning,
transfer and expression of genes in vitro, ex vivo or in vivo. Such
genes of interest are, for example, genes encoding enzymes, blood
derivatives, hormones, lymphokines: interleukins, interferons, TNF,
and the like (FR 9203120), growth factors, neurotransmitters or
their precursors of synthesis enzymes, trophic factors: BDNF, CNTF,
NGF, IGF, GMF, aFGF, bFGF, NT3, NT5, and the like, apolipo
proteins: ApoAI, ApoAIV, ApoE, and the like (WO94/25073),
dystrophin or a minidystrophin (WO93/06223), tumour suppressor
genes: p53, Rb, Rap1A, DCC, k-rev, and the like (WO94/24297), genes
encoding factors involved in clotting: Factors VII, VIII, IX and
the like, suicide genes: thymidine kinase, cytosin deaminase and
the like, or all or part of a natural or artificial immunoglobulin
(Fab, ScFv, and the like, WO94/29446), and the like. The gene of
interest may also be a gene or an antisense sequence, whose
expression in the target cell makes it possible to control the
expression of genes or the transcription of cellular mRNAs. Such
sequences may, for example, be transcribed, in the target cell,
into RNAs complementary to cellular mRNAs and thus block their
translation into protein, according to the technique described in
Patent EP 140 308. The gene of interest may also be a gene encoding
an antigenic peptide, capable of generating an immune response, for
the production of vaccines. It may be in particular antigenic
peptides specific for the Epstein-Barr virus, the HIV virus, the
hepatitis B virus (EP 185 573), the pseudorabies virus, or specific
for tumours (EP 259 212). The gene may be any DNA (gDNA, cDNA and
the like) encoding a product of interest, potentially including the
appropriate expression signals (promoter, terminator and the
like).
[0089] These viruses may be used in vitro for the production of
these recombinant proteins. They may also be used, still in vitro,
to study the mechanism of action of these proteins or to study the
regulation of the expression of genes or the activity of
promoters.
[0090] They may also be used in vivo, for the creation of animal
models or of transgenic animals. They may also be used for the
transfer and expression of genes in vivo, in animals or man, in
gene or cell therapy procedures.
[0091] The present application will be described in greater detail
with the aid of the following examples which should be considered
as illustrative and nonlimiting.
Legend to the Figures
[0092] FIG. 1: Schematic representation of the production of a
third-generation defective recombinant adenovirus (defective for
the E1 and E4 functions) using a baculo-E1, E4.
[0093] FIGS. 2-4: Schematic representation of the production of a
minimum adenovirus using a first baculovirus to introduce the
defective adenoviral genome and a second baculovirus to introduce
the compleentation functions.
[0094] FIG. 5: Schematic representation of the production of a
recombinant AAV defective for the Rep and Cap functions using a
baculo-Rep/Cap.
EXAMPLES
[0095] 1. Cells Used
[0096] The cells used within the framework of the invention may be
obtained from any cell line or population capable of being infected
by an adenovirus or an AAV or a retrovirus or by a baculovirus,
compatible with a use for therapeutic purposes. It is more
preferably a mammalian, particularly human, cell. There may be
mentioned more particularly:
The Cells of the 293 Line:
[0097] The 293 line is a human embryonic kidney cell line
containing the left end (about 11-12%) of the genome of the
serotype 5 adenovirus (Ad5), comprising the left ITR, the
encapsidation region, the E1 region, including E1a, E1b, the region
encoding the pIX protein and part of the region encoding the pIVa2
protein (Graham et al., J. Gen. Virol. 36 (1977) 59). This line is
capable of transcomplementing recombinant adenoviruses defective
for the E1 region, that is to say lacking all or part of the E1
region, and of producing viral stocks having high titres.
The Cells of the A549 Line
[0098] Cells complementing the E1 region of the adenovirus were
constructed from A549 cells (Imler et al., Gene Ther. (1966) 75).
These cells contain a restricted fragment of the E1 region, lacking
the left ITR, placed under the control of an inducible
promoter.
The Cells of the HER Line
[0099] The human embryonic retinal (HER) cells can be infected with
an adenovirus (Byrd et al., Oncogene 2 (1988) 477). Adenovirus
encapsidation cells prepared from these cells have been described
for example in application WO94/28152 or in the article by Fallaux
et al. (Hum. Gene Ther. (1996) 215). There may be mentioned more
particularly the 911 line comprising the E1 region of the genome of
the Ad5 adenovirus, from nucleotide 79 to nucleotide 5789,
integrated into the genome of HER cells. This cell line allows the
production of viruses defective for the E1 region.
The IGRP2 Cells
[0100] The IGRP2 cells are cells obtained from cells 293, by
integration of a functional unit of the E4 region under the control
of an inducible promoter. These cells allow the production of
viruses defective for the E1 and E4 regions (Yeh et al., J. Virol
(1966) 70).
The VK Cells
[0101] The VK cells (VK2-20 and VK10-9) are cells obtained from
cells 293, by integration of the entire E4 region under the control
of an inducible promoter, and the region encoding the protein pIX.
These cells allow the production of viruses defective for the E1
and E4 regions (Krougliak et al., Hum. Gene Ther. 6 (1995)
1575).
The 293E4 Cells
[0102] The 293E4 cells are cells obtained from cells 293, by
integration of the entire E4 region. These cells allow the
production of viruses defective for the E1 and E4 regions
(WO95/02697; Cancer Gene Ther. (1995) 322).
[0103] The Sf9 and Sf21 cells are embryonic Lepidoptera cells.
These cells are accessible in collections (No. CRL-1711 ATCC) as
well as their culture conditions. They are also commercially
available (Gibco). See also King and Possee: The baculovirus
expression system, London: Chapman and Hall, 1992.
The Human Hepatocytic Cells
[0104] The HepG2 and Hep3B and HuH7 cells are human lines derived
from hepatocarcinomas. They are accessible in depository
collections and their properties have been described for example in
Patents U.S. Pat. No. 4,393,133 and 4,393,133.
[0105] Human cell line KB: Derived from a human epidermal
carcinoma, this line is accessible at the ATCC (ref. CCL17) as well
as the conditions allowing its culture.
[0106] Human cell line Hela: derived from a carcinoma of the human
epithelium, this line is accessible at the ATCC (ref. CCL2) as well
as the conditions allowing its culture.
[0107] Cell line W162: These cells are Vero cells comprising,
integrated into their genome, the E4 region of the Ad2 adenovirus.
These cells have been described by Weinberg et al., (PNAS 80 (1983)
5383).
[0108] 2. Infection of Human Cells with a Recombinant
Baculovirus
[0109] This example describes the capacity of baculoviruses to
infect cells of human origin.
[0110] Human cells (particularly 293 or derivatives thereof) are
infected with various dilutions of a solution of recombinant
baculovirus expressing the LacZ gene under the control of the RSV
LTR. 48 hours after infection, the appearance of blue cells is
revealed, demonstrating the infectability of these cells by a
baculovirus.
[0111] 3. Construction of Baculoviruses Expressing the E1 Region of
the Adenovirus and of a Corresponding Defective Adenovirus
[0112] 3-1 Cloning of Two Cassettes for the Expression of E1
[0113] The plasmid AE2 is obtained from the cloning, in PCRII
(Invitrogen) of the product of the PCR performed on pBRE1 with the
oligonucleotides 5'-TCCTTGCATTTGGGTAACAG-3' and
5,'-GCGGCCGCTCAATCTGTATCTTC-3'; this PCR product contains
nucleotides 3198 to 3511 of Ad5, that is to say the 3' end of the
E1B region. The plasmid pBRE1 contains nucleotides 1 to 5788 of Ad5
cloned into pBR322, deleted roughly of nucleotides 1300 to
2300.
[0114] The plasmid AE3 is derived from the cloning of the NotI-KpnI
fragment of AE2, containing the PCR product, into pCDNA3
(Invitrogen) digested with NotI-KpnI. It contains nucleotides 3198
to 3511 of Ad5 followed by the polyadenylation site of BGH.
[0115] The plasmid AE4 is derived from the cloning of the
BglII-PvuII fragment of AE3 into pBRE1. AE4 is a plasmid containing
the following E1 expression cassette:
[0116] nucleotides 1 to 3511 of Ad5, that is to say left ITR,
encapsidation sequences, E1A promoter, E1A gene, E1B promoter, E1B
gene
[0117] the polyA of the bovine growth hormone (BGH) obtained from
pCDNA3.
[0118] pBRE1 was digested with BstNI, and then digested with T4 DNA
polymerase in order to fill the protruding 5' end, and then
digested with XbaI. The fragment thus generated containing
nucleotides 391 to 1339 of Ad5 was introduced into pic20H digested
with SmaI-XbaI (nonmethylated site), to give the plasmid AE5.
[0119] The plasmid AE6 is derived from the cloning of the
EcoRI-SmaI fragment of AE5 into AE4 digested with EcoRI-SmaI. AE6
is a plasmid containing the following E1 expression cassette:
[0120] nucleotides 391 to 3511 of Ad5, that is to say "reduced"
promoter of E1A, E1A gene, E1B promoter, E1B gene,
[0121] the polyA of the bovine growth hormone (BGH) obtained from
pCDNA3.
[0122] 3-2 Cloning of these Two E1 Cassettes into a Baculovirus
[0123] The EcoRI-SphI fragments (SphI protruding 5' end which has
been made blunt beforehand by digestion with T4 DNA polymerase) of
the plasmids AE4 and AE6 are cloned into the plasmid pAcSG2
(Pharmingen) between the EcoRI and EcoRV sites. This generates the
plasmids AE14 and AE15 respectively. In both cases, the E1 region
is introduced into the locus of the polyhedrin gene (polyhedrin
gene+polyhedrin promoter being deleted).
[0124] The plasmids AE14 and 15 are cotransfected with the DNA of
the baculovirus BaculoGold derived from the AcNPV strain
(Pharmingen) into Sf9 insect cells, in order to generate the two
corresponding recombinant baculoviruses BacAE14 and BacAE15,
carrying the two cassettes for the expression of E1 integrated into
the locus of the polyhedrin gene.
[0125] 3-3 Cloning of a recombinant Adenovirus Carrying a New E1
Deletion
[0126] The plasmid pCO1 (WO96/10088) was digested with BstXI, and
then digested with T4 DNA polymerase in order to remove the
protruding 3' end, and then digested with RsaI. The fragment thus
generated containing nucleotides 3513 to 4607 of Ad5 was introduced
into pBS-SX+ (Stratagene) linearized with EcoRV and then digested
with calf alkaline phosphatase, to generate the plasmid AE0.
[0127] The plasmid pMA37 is obtained by ligation of the
fragments:
[0128] EcoRV-NsiI of pXL2756, containing sacB. pXL2756 possesses
the counter-selection gene SacB free of its EcoRI and KpnI sites,
the kanamycin resistance gene, a multiple cloning site and a
replication origin ColE1,
[0129] NdeI-NsiI of pCO1 (containing the adeno sequences)
[0130] SalI (protruding 5' end filled with T4 DNA polymerase)-AseI
of pXL2756 (kanamycin-resistant vector).
[0131] pMA37 therefore contains:
[0132] the kanamycin resistance gene
[0133] the SacB gene conferring sucrose sensitivity on bacteria
expressing it
[0134] sequences 1 to 382 (HinfI) of Ad5 followed by sequences 3446
to 4415 (NsiI) of Ad5; there is no transgene.
[0135] The plasmid AE11 was constructed by introducing the
XhoI-NsiI fragment of AE0 into pMA37 digested with SalI-NSiI. It
thus contains:
[0136] the kanamycin resistance gene
[0137] the SacB gene conferring sucrose sensitivity on bacteria
expressing it,
[0138] sequences 1 to 382 (HinfI) of Ad5 followed by sequences 3513
to 4415 (NsiI) of Ad5; there is no transgene.
[0139] From the plasmid AE11 are then constructed the suicide
shuttle vectors (by insertion of the transgene of interest)
allowing the construction of recombinant adenoviruses by
recombination in E. Coli. AE11 contains no sequence homologous with
the plasmid AE6. The plasmids AE11 and AE4 have in common sequences
1 to 382 (HinfI) of Ad5 upstream of E1A, but there is no homology
downstream of the E1 cassette between these two plasmids. Thus,
there can be no generation of RCA by homologous recombination
between an adeno carrying the E1 deletion existing in AE11 (that is
to say 382-3513) and the baculoviruses BacAE14 or BacAE15.
[0140] 3-4 Construction of a First-generation Recombinant
Adenovirus
[0141] In the first instance, a stock of BacAE14 or 15 is prepared
according to conventional techniques. Next, the competent cells
(for example HuH7) are transfected with 5 .mu.g of plasmid pXL2822
digested with PacI (the plasmid pXL2822 contains all the Ad5
deleted for E1 (382-3446 or 382-3513) and E3 (28592-30470) and
carries a cassette CMV-.beta.Gal), and infected, simultaneously or
otherwise, at an MOI between 10 and 1000, with BacAE14 or 15. When
the cells are lysed, the transfection supernatant is harvested, and
then applied onto "fresh" competent cells previously or
simultaneously infected with BacAE14 or 15 (MOI 10 to 1000), so as
to amplify the adenovirus Ad2822, and so on until a stock of Ad2822
is obtained. The monitoring of the amplification of Ad2822 is
facilitated by the presence of lacZ in this virus. On each
amplification, the supernatant is thus pseudotitrated on W162. The
genome of the virus is analysed during amplifications so as to
check its integrity. Finally, this strategy has the advantage of
not generating RCA in the adenovirus stocks thus produced. This
absence of contamination is also verified.
[0142] 4. Construction of a Baculovirus Expressing the E1 and E4
Regions of the Adenovirus
[0143] 4-1 Construction of the Baculovirus E1,E4 (Bac.E1-E4)
[0144] The protocol used is the following: the E1 and E4 regions
are cloned in reverse orientation to the locus of the polyhedrin
(Ph) gene, into the shuttle vector pBacPAK8 (Clontech, USA) to give
pBacE1-E4. The recombinant baculovirus is then isolated according
to conventional techniques by cotransfection of pBacE1-E4 and of
the DNA of the baculovirus BacPAK6 into Sf9 cells (Kitts and
Possee, Biotechniques 14(5) (1993) 810). The presence of E1 and E4
in the genome of the recombinant baculoviruses is then checked by
PCR using infected Sf9 cells. The transcription of E1 and E4 in the
Huh7 cells infected with the purified recombinant baculovirus is
analysed by RT-PCR using cytoplasmic RNAs.
[0145] For the construction of the E1-E4 baculovirus, various
fragments carrying E1 may be introduced. The fragments used in this
example are those described in Example 3 for the construction of
the Baculovirus-E1, containing the E1 regions under the control of
their own promoter (reduced E1a promoter and E1b promoter). The E4
fragments used are the following:
[0146] fragment MaeII-MscI 32720-35835 carrying the entire E4
[0147] fragment BglII-PvuII 34115-33126 carrying the frame ORF6
[0148] fragment BglII-BglII 34115-32490 carrying the frames
ORF6-ORF6/7
[0149] fragment PvuII-AluI 34801-334329 carrying the frame ORF3
[0150] These fragments are placed alternatively under the control
of the E4 promoter or of different promoters, particularly of the
HSV-TK or CMV promoter or of the RSV-LTR.
[0151] The positions given above refer to the sequence of the
wild-type Ad5 adenovirus as published and accessible on database.
Although some minor variations may exist between the various
adenovirus serotypes, these positions are generally transposable to
other serotypes, and in particular to Ad2, Ad7, Ad12 and Ad
CAV-2.
[0152] 4-2 Transcomplementation of Ad.DELTA.E1.DELTA.E4-LacZ with
Bac.E1-E4
[0153] The production of the adenovirus Ad.DELTA.E1.DELTA.E4-LacZ
is obtained by introduction into the competent cells of the E1, E4
baculovirus prepared in Example 4-1 and of the adenoviral genome
defective for E1 and E4 (see FIG. 1). The optimum conditions for
the production of Ad.DELTA.E1.DELTA.E4-LacZ in the competent cells,
by transcomplementation with the purified Bac.E1-E4, are analysed.
The Ad.DELTA.E1.DELTA.E4 titre is determined as number of
.beta.-galactosidase transduction units (t.d.u.) in the W162 line
and the transcomplementation efficiency obtained is compared with
that of the encapsidation line IGRP2.
[0154] 5. Construction of a Baculovirus Complementing the Whole of
the Adenovirus Genome
[0155] Although the simultaneous expression of several proteins,
which may represent up to 13 kb of sequence, from a single virus
has been reported (Belyaev and Roy, NAR 21 (1993) 1219), the
maximum cloning capacity of the baculovirus is not highly
documented. This example now shows that it is possible to clone the
adenovirus genome deleted of its encapsidation signal (Ad.Psi-) and
bordered by two loxP sites [loxP-ITR-ITR to E4 -loxP] into the
baculovirus. The infection of competent cells expressing the
recombinase Cre, in an inducible manner or otherwise (Cre or Cre-ER
line, see Example 7), with this recombinant baculovirus and the
activation of the recombinase Cre, allow the excision and the
circularization of the adenoviral genome. The latter is then
capable of transducing the early genes, of replicating and of
activating the late genes, but incapable of being encapsidated, and
thus serves as helper for the production of a minimum adenovirus
(see FIGS. 2-4). In this system, the production of the minimum
adenoviruses is based on the co-infection by two baculoviruses and
the realization of 2 recombination events between the loxP sites.
This approach has the advantage of not generating adenoviral
particles from the helper virus.
[0156] The construction of the Ad.Psi-genome is carried out in E.
coli. For that, the complete genome of the Ad5 adenovirus is cloned
into a prokaryotic cloning vector, ITRs attached. The Psi sequence
is deleted by enzymatic cleavage and ligation, or by site-directed
mutagenesis. A LoxP sequence is then introduced on either side of
the adenoviral genome, in parallel orientation. The resulting
construct [loxP-ITR-ITR, .DELTA.Psi to E4-loxp] is then cloned into
a shuttle vector allowing recombination with a baculovirus,
according to the strategy described in Example 3. The recombinant
baculovirus obtained, called BacAd.Psi-, is then isolated according
to conventional methods.
[0157] 6. Construction of a Baculovirus Comprising the Genome of
the Defective Recombinant Adenovirus in Excisable Form
[0158] This example describes the construction of a baculovirus
which makes it possible to provide in the competent cells the
genome of the defective recombinant adenovirus. More particularly,
the recombinant adenovirus is defective for the whole of the coding
regions, and retains only the ITR and Psi regions (minimum
adenovirus, or Ad.DELTA.).
[0159] 6-1 Construction of a Minigenome (Ad.DELTA.) in E. Coli
[0160] A plasmid p[loxP-(ITR-ITR-Psi-P.CMV-LacZ-pA)-loxP] is
constructed. For that, a copy of the ITR sequence of the adenovirus
is isolated by enzymatic cleavage and/or amplified by PCR, and then
cloned upstream of the ITR-Psi sequence contained in the shuttle
vector of the adenovirus pGY63. This vector is derived from pCO1
(WO96/10088) and possesses the LacZ gene under the control of the
immediate-early promoter of the cytomegalovirus (P.CMV) ending with
the polyadenylation signal of the SV40 virus (pA), cloned between
the ITR-Psi sequence and the gene encoding pIX. The region
(ITR-ITR-Psi-P.CMV-LacZ-pA) of this vector (corresponding to a
minimum adenovirus genome) is then isolated by enzymatic cleavage
and cloned between the LoxP sites into the multiple cloning site of
the plasmid pBS246 (Gibco), to generate the plasmid
p[loxP-(ITR-ITR-Psi-P.CMV-LacZ-pA)-loxP]. The capacity to produce
adEnovirus minigenomes from a circular DNA and to encapsidate them
is then tested by transfection of this plasmid into the IGRP2 line
infected with Ad..DELTA.E1.DELTA.E4 expressing the recombinase Cre
(AdCre). The minimum adenoviruses are amplified by a few successive
passages of the supernatant from the transfection in the IGRP2
line. They are then purified by isopycnic caesium chloride gradient
centrifugation and quantified by pseudotitration in the W162 line.
It is understood that the LacZ gene can be easily replaced by any
other nucleic acid of interest, by conventional molecular biology
techniques.
[0161] 6.2 Cloning of an Excisable Minigenome into a
Baculovirus
[0162] The construct carrying the minigenome Ad.DELTA. bordered by
the two loxP sites described above is cloned at the P10 locus of
the baculovirus into the shuttle vector pAcUW1 (Pharmingen, USA).
The baculovirus Bac.Ad.DELTA. is then produced and isolated by
conventional techniques of cotransfection into the abovementioned
Sf9 cells, and selected by its phage phenotype (white), after
staining with X-Gal. This baculovirus therefore carries a highly
defective adenoviral genome, flanked by two loxP regions in direct
orientation.
[0163] 6-3 Production of Ad.DELTA. by Transcouplementation with the
Baculovirus BacAdPsi-
[0164] Competent cells are simultaneously co-infected with the
baculovirus BacAdPsi- (described in Example 5), carrying the
transcomplementation functions of the whole of the adenoviral
genome, and with the baculovirus Bac.Ad.DELTA. carrying the genome
of the PseudoAdenovirus (described above). The recombinase Cre is
provided either by adding the protein into the culture medium, or
by transfecting the cells with a plasmid or a virus (baculovirus)
expressing Cre, or by expression of a cassettee stably integrated
into the genome of the line (as described in Example 7). The
minimum adenovirus is amplified by successive passages of the
culture supernatants of cells co-infected with BacAd.Psi- and with
the supernatant, and then purified and titrated according to the
techniques mentioned above. This technique makes it possible to
obtain Ad.DELTA. as sole virus, which allows its isolation and its
purification by conventional techniques. In addition, the titres
obtained are compatible with an industrial use.
[0165] 7. Construction of a Line Expressing the Cre Protein
[0166] A line expressing Cre, in an inducible manner or otherwise,
is constructed in order to increase the efficiency of recombination
between the loxP sites in the baculovirus of the invention (for
example Bac.Ad.DELTA. and BacAd.Psi-) and to control the expression
of Cre. In this construct, Cre is expressed alone or in the form of
a C-terminal fusion protein with the oestradiol receptor (ER)
binding domain (Metzger et al., 1996, cited above), under the
control of a ubiquitous promoter, preferably a strong promoter
inducible or otherwise. More particularly, the promoters used are
the pGRE5 promoter, the metallothionin promoter, the SV40 promoter
or the promoter of the HSV-TK gene.
[0167] To construct these Cre lines, the compEtent cells are
cotransfected with two plasmids, one containing the Cre expression
cassette (Cre or Cre-ER) and the other that for a selectable marker
(Neo). G418-resistant clones are selected, the Cre activity in
these clones is tested by transfection of the plasmid
p(P.CMV-loxP-ATG-stop-pA-LoxP-LacZ). This plasmid contains the LacZ
gene inactivated by introducing between the promoter (P.CMV) and
the beginning of LacZ a succession of stop codons in the three
reading frames and the signal for termination of transcription and
the polyadenylation signal of the SV40 virus, bordered by two loxP
sites. The expression of Cre in the clones, in the presence or
otherwise of the inducer (oestradiol), is then revealed by the
.beta.-galactosidase activity induced by recombination between the
two loxP sites. Several clones stably expressing the fusion protein
Cre-ER or the protein Cre alone from the promoters and the
competent cells specified below are thus selected. These clones can
be used for the production of viruses according to the
invention.
1 HSV-TK SV40 MMTV GRE5 Competent promoter promoter promoter
promoter cell Cre- Cre- Cre- Cre- Recombinase Cre ER Cre ER Cre ER
Cre ER 293 #1 #7 #13 #19 #25 #31 #37 #43 IGRP2 #2 #8 #14 #20 #26
#32 #38 #44 Huf7 #3 #9 #15 #21 #27 #33 #39 #45 HepG2 #4 #10 #16 #22
#28 #34 #40 #46 HER #5 #11 #17 #23 #29 #35 #41 #47 Vero #6 #12 #18
#24 #30 #36 #42 #48
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