U.S. patent application number 09/810490 was filed with the patent office on 2003-10-30 for viral vectors and line for gene therapy.
Invention is credited to Lusky, Monika, Mehtali, Majid, Rittner, Karola.
Application Number | 20030203488 09/810490 |
Document ID | / |
Family ID | 26232115 |
Filed Date | 2003-10-30 |
United States Patent
Application |
20030203488 |
Kind Code |
A1 |
Mehtali, Majid ; et
al. |
October 30, 2003 |
Viral vectors and line for gene therapy
Abstract
Novel viral vectors in which the expression of viral genes is
regulated in such a way that it is functional in a complementation
cell and non-functional in a host cell, as well as viral particles
and host cells containing said novel vectors, are disclosed. A
complementation cell including a viral gene expression regulator,
and a method for preparing infectious viral particles, are also
disclosed. Finally, a pharmaceutical composition containing said
vectors, and the therapeutical use thereof, are disclosed.
Inventors: |
Mehtali, Majid;
(Illkirch-Graffenstaden, FR) ; Lusky, Monika;
(Freiburg, DE) ; Rittner, Karola; (Strasbourg,
FR) |
Correspondence
Address: |
BURNS, DOANE, SWECKER & MATHIS, L.L.P.
P.O. Box 1404
Alexandria
VA
22313-1404
US
|
Family ID: |
26232115 |
Appl. No.: |
09/810490 |
Filed: |
March 19, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09810490 |
Mar 19, 2001 |
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08809562 |
Mar 31, 1997 |
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6204060 |
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08809562 |
Mar 31, 1997 |
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PCT/FR96/01165 |
Jul 24, 1996 |
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Current U.S.
Class: |
435/456 ;
435/235.1 |
Current CPC
Class: |
A61P 35/00 20180101;
C12N 2840/20 20130101; A61P 3/10 20180101; A61P 7/04 20180101; A61P
21/00 20180101; C12N 2710/10343 20130101; A61P 31/12 20180101; A61P
43/00 20180101; C12N 7/00 20130101; C12N 2830/006 20130101; C12N
15/86 20130101; C12N 2710/10352 20130101; A61K 48/00 20130101; A61P
31/18 20180101 |
Class at
Publication: |
435/456 ;
435/235.1 |
International
Class: |
C12N 015/86; C12N
007/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 24, 1995 |
FR |
95 08946 |
Apr 9, 1996 |
FR |
96 04413 |
Claims
1. Viral vector, characterized in that it compris s an expression
unit containing one or mor viral genes said expression unit being
functional in a complementation cell and nonfunctional in a host
call, and comprising one or more heterologous regulator
sequence(s).
2. Viral vector according to claim 1, characterized in that said
expression unit comprises one or more regulatory sequence(s)
enabling to activate the expression of said viral gene in the
presence of an inducer and/or to inhibit the expression of said
viral gene in the presence of a repressor.
3. Viral vector according to claim 1 or 2, characterized in that
said regulatory sequence can act at the level of transcription,
elongation, transport or stability of the messenger or
translation.
4. Viral vector according to claim 3, characterized in that said
regulatory sequence in placed at the level of the promoter of said
unit, and more *specially up-stream of the TATA box.
5. Viral vector according to one of claims 1 to 4, characterized in
that said expression unit comprises one or more regulatory
sequence(s) selected from the TAR, RRE, GRE, PRE, ERE and Gal4 UAS
sequences and the regulatory sequences of the metallothionein gone
and of the bacterial tryptophans lactose and tetracycline
operons.
6. Viral vector according to claim 5, characterized in that said
expression unit comprises one or more regulatory sequence(s)
derived from the tetracycline operon, placed upstream of the TATA
box of said promoter, to give a promoter which in activable by an
inducer of the tetracycline transactivator (tTA) type and
repressible by tetracycline.
7. Viral vector according to claim 5, characterized in that said
expression unit comprises one or more regulatory sequence(s)
derived from the tetracycline operon, placed downstream of the TATA
box of said promoter, to give a promoter which is r pressible by
the tetracycline repressor (T tR).
8. Viral vector according to one of claims 1 to 7, derived from a
virus selected from the herpesvirus, cytomegalovirus, AAV
(adeno-associated virus), poxvirus and adenovirus.
9. Adenoviral vector according to claim 8, derived from an
adenovirus of human, canine, avian, bovine, murine, ovine, porcine
or simian origin, or alternatively from a hybrid comprising
adenoviral genome fragments of different origins.
10. Viral vector according to claim 8 or 9, characterized in that
it is defective for replication.
11. Adenoviral vector according to claim 10, characterized in that
it lacks at least all or part of the E1 region and, optionally, all
or part of the E3 region.
12. Adenoviral vector according to one of claims 9 to 11,
characterized in that it comprises one or more expression unit(s)
containing one or more viral genes of the E2, E4 or L1-L5
regions.
13. Adenoviral vector according to claim 12, characterized in that
it comprises an expression unit containing one or more regulatory
sequence(s) derived from the tetracycline operon, placed upstream
of the TATA box of the promoter and open reading frames (ORFs) 6
and 7 of the E4 region, so that the expression of said reading
frames is activable by an inducer of the tetracycline
transactivator (tTA) type and repressible by tetracycline.
14. Viral vector according to one of claims 1 to 13, characterized
in that it comprises an exogenous nucleotide sequence placed under
the control of the elements needed for its expression in the host
cell.
15. Viral vector according to claim 14, characterized in that the
exogenous nucleotide sequence is selected from the genes coding for
a cytokine, a cell or nuclear receptor, a ligand, a coagulation
factor, the CFTR protein, insulin, dystrophin, a growth hormone, an
enzyme, an enzyme inhibitor, a polypeptide having an antitumor
effect, a polypeptide capable of inhibiting a bacterial, parasitic
or viral infection, and in particular HIV, an antibody, a toxin, an
immunotoxin and a marker.
16. Infectious viral particle comprising a viral vector according
to one of claims 1 to 15.
17. Eukaryotic host cell comprising a viral vector according to one
of claims 1 to 15 or an infectious viral particle according to
claim 16.
18. Complementation cell, characterized in that it comprises an
inducer and/or a repressor.
19. Complementation cell according to claim 18, characterized in
that it comprises a DNA fragment coding for an inducer and/or a
repressor.
20. Complementation cell according to claim 18 or 19, characterized
in that it is derived from line 293.
21. Complementation call according to one of claims 18 to 20, for
the complementation of an adenoviral vector which is defective for
the E1 function and at least one second, late or early adenoviral
function, characterized in that it comprises: (i) a first cassette
for the expression of all or part of the E1 region of an
adenovirus, placed under the control of the elements necessary for
its expression in said complementation cell, and (ii) a second
cassette for the expression of all or part of a late or early
region of an adenovirus other than the E1 region, placed under the
control of the elements necessary for its expression in said
complementation cell, said elements comprising one or more
regulatory sequences according to claims 5 to 7.
22. Complementation cell according to claim 21, characterized in
that said elements of the second expression cassette comprise a
minimal promoter equipped at its 5' end with 1 to 20 tet O
sequences.
23. Complementation cell according to claim 22, characterized in
that said elements of the second expression cassette comprise a
minimal promoter derived from the CMV virus (cytomegalovirus)
equipped at its 5' and with 7 tet O sequences.
24. Complementation cell according to one of claims 18 to 23 for
the complementation of an adenoviral vector which is defective for
the E1 and E4 functions, characterized in that the second
expression cassette in a cassette for the expression of all or part
of the E4 region of an adenovirus.
25. Complementation cell according to claim 24, characterized in
that said second expression cassette is a cassette for the
expression of the sequences coding for open reading frames 6 and 7
(ORFs 6/7) of the E4 region of an adenovirus.
26. Complementation cell according to one of claims 18 to 23 for
the complementation of an adenoviral vector which is defective for
the E1 and E2 functions, characterized in that said second
expression cassette is a cassette for the expression of all or part
of the E2 region of an adenovirus.
27. complementation cell according to claim 26, characterized in
that said second expression cassette is a cassette for the
expression of the sequences coding for the DBP protein (DNA binding
protein) of the E2 region of an adenovirus.
28. Complementation cell according to claim 26, characterized in
that said second expression cassette is a cassette for the
expression of the sequences coding for a temperature-sensitive
mutant of the DBP protein of the E2 region of an adenovirus.
29. Complementation cell according to one of claims 18 to 28 for
the complementation of an adenoviral vector which is defective for
the E1 function and at least two other, late or early adenoviral
functions, characterized. in that it comprises, in addition, a
third cassette for the expression of all or part of a late or early
region of an adenovirus other than the E1 region and the adenoviral
region of the second expression cassette, placed under the control
of the elements necessary for its expression in said
complementation cell, and preferably of a promoter as defined in
claim 5, 6 or 7.
30. complementation cell according to claim 29 for the
complementation of an adenoviral vector which is defective for the
E1, E2 and E4 functions, comprising: (i) a first cassette for the
expression of all or part of the E1 region of an adenovirus, placed
under the control of the elements necessary for its expression in
said complementation cell, (ii) a second cassette for the
expression of all or part of the E4 region of an adenovirus, placed
under the control of the elements necessary for its expression in
said complementation cell, and (iii) a third cassette for the
expression of all or part of the E2 region of an adenovirus, placed
under the control of the elements necessary for its expression in
said complementation call, said elements of the second and/or third
expression cassette comprising a promoter equipped with at least
one tet O sequence, and more especially a minimal promoter derived
from the C virus (cytomegalovirus), equipped. at its 5' end with 7
tet O sequences.
31. Complementation cell according to one of claims 18 to 30,
characterized in that the titer of viral particles produced by said
complementation cell is greater than 5.times.10.sup.8 pfu (plaque
forming units)/ml.
32. Complementation cell according to one of claims 18 to 31,
characterized in that the titer of viral particles produced by said
complementation cell is greater than 1.times.10.sup.10 ifu
(infectious units)/ml.
33. Method of preparation of an infectious viral particle according
to claim 16, according to which: (i) a viral vector according to
one of claims 1 to 15 in introduced into a complementation call
capable of complementing in trans said viral vector, to obtain a
transfected complementation cell; (ii) said transfected
complementation cell is cultured under suitable conditions to
permit the expression of the viral genes and the production of said
infectious viral particle; and (iii) said infectious viral particle
is recovered in the cell culture.
34. Method according to claim 33, characterized in that said viral
vector in an adenoviral vector and said complementation cell is
according to claim 20.
35. Method of preparation of an infectious adenoviral particle,
according to which: (i) an adenoviral vector is introduced into a
complementation cell according to one of claims 21 to 32, to obtain
an infected complementation cell, (ii) said transfected [sic]
complementation cell is cultured under suitable conditions to
permit the expression of the viral genes and the production of said
infectious viral particle; and (iii) said infectious viral particle
is recovered in the cell culture.
36. Pharmaceutical composition comprising a viral vector according
to one of claims 1 to 15, an infectious viral particle according to
claim 16 or obtained employing a preparation method according to
one of claims 33 to 35, a eukaryotic host call according to claim
17 or a complementation cell according to one of claims 18 to 32,
in combination with a vehicle which is acceptable from a
pharmaceutical standpoint.
37. Therapeutic or prophylactic use of a viral vector according to
one of claims 1 to 15, of an infectious viral particle according to
claim 16 or obtained employing a preparation method according to
one of claims 33 to 35, of a eukaryotic host call according to
claim 17 or of a complementation cell according to one of claims 18
to 32, for the preparation of a medicinal product intended for the
treatment of the human or animal body by gone therapy.
38. Use according to claim 37, in combination with a repressor.
Description
[0001] The present invention relates to new viral vectors
permitting the transfer and expression of genes of interest in a
host cell or body, the expression of the viral genes being
regulated so as to be functional in a complementation cell and
nonfunctional in the host cell or body. It also relates to the
cells containing these now vectors, as well as to a method for
preparing infectious viral particles intended for therapeutic use.
The invention in of very special interest in relation to prospects
for gene therapy, in particular in man.
[0002] The possibility of treating human diseases by gene therapy
has changed in a few years from the stage of theoretical
considerations to that of clinical applications. The first protocol
applied to man was initiated in the US in September 1990 on a
patient who was genetically immunodeficient as a result of a
mutation affecting the gene coding for adenine deaminase (ADA). The
relative success of this first experiment encouraged the
development of now gene therapy protocols for various genetic or
acquired diseases (infectious diseases, and viral diseases in
particular, such as AIDS, or cancers). The large majority of the
protocols described hitherto employ viral vectors to transfer the
therapeutic gene to the cells to be treated and to express it
therein.
[0003] To date, retroviral vectors are among the ones most widely
used on account of the simplicity of their gene. . or, &part
from their restricted capacity for cloning, they present two major
drawbacks which limit their systematic use: on the one hand they
chiefly infect dividing cells, and on the other hand, an a result
of their integration at random in the genome of the. host cell, the
risk of insertional mutagenesis is not insignificant. For this
reason, many scientific teams have endeavored to develop other
types of vector, among which those originating from adenoviruses,
adeno-associated viruses (AAV), cytomegaviruses, poxviruses and
herpesviruses may be mentioned. Generally speaking, their
organization and their infection cycle are amply described in the
literature available to a person skilled in the art.
[0004] In this connection, the use of adenoviral vectors has8 been
seen to be a prosing alternative. Adenoviruses have been
demonstrated in many animal species, have a broad host range, have
little pathogenicity and do not present the drawbacks associated
with retroviruses since they are nonintegrative and replicate also
in resting cells. As a guide, their genome consists of a linear,
double-stranded DNA molecule of approximately 36 kb carrying more
than about thirty genes, both early genes necessary for viral
replication and late structural genes (see FIG. 1).
[0005] The early genes are divided into 4 regions dispersed in the
adenoviral genome (B1 to E4; E standing for early). They contain 6
transcription units which possess their own promoters. The late
genes (L1 to L5; L standing for late) partially overlap the early
transcription units and are, for the most part, transcribed from
the major late promoter (MLP).
[0006] At the present time, all the adenoviral vectors used in gone
therapy protocols lack most of the E1 region essential for
replication, in order to avoid their dissemination in the
environment and the host body. Some of then contain additional
deletions, in particular in the nonessential E3 region, enabling
their cloning capacity to be increased. The genes of interest are
introduced into the viral DNA in place of one or other deleted
region. Deletion of the E1 region renders the viral genome
deficient for replication. However, E1.sup.31 viruses my be
propagated in a complementation call line, which supplies in trans
the deleted viral functions to generate an infectious viral
particle. Line 293, established from human embryonic kidney cells,
which complements the E1 function effectively (Graham et al., 1977,
J. Gen. Virol. 36, 59-72), is commonly used. The E3 region is
nonessential and does not need to be complemented.
[0007] While the feasibility of gone transfer using these first
generation vectors is now well established, the question of their
safety remains unresolved. Apart from the safety aspects (risk of
generating RCAs, that is to say replication competent particles),
the problem of their toxicity arises. in effect, the first clinical
trials have revealed the induction of inflammatory responses
associated with the expression of the viral genes in the host.
[0008] Second generation adenoviral vectors have recently been
proposed in the literature. They retain the In cis regions
necessary for replication of the virus in the infected cell (ITRs
and encapsidation sequences) and contain substantial internal
deletions aimed at abolishing the bulk of the viral genes whose
expression in vivo is not desirable. However, these vectors of the
prior art have some drawbacks which limit their exploitation at an
industrial level. It in, in effect, necessary to have at one's
disposal now lines complementing the collective deleted functions
and enabling viral particles to be produced at a high titer. In
point of fact, such a line, in order to be optimal in term of
capacity for growth and yield of viral particles, in specially
difficult to generate on account of the cytotoxicity of the
adenoviral genes.
[0009] The present invention enables these drawbacks to be
remedied. On the one hand, a new line derived from line 293
complementing the E1 and E2 or E4 adenoviral functions, for the
amplification of conventional second generation adenoviral vectors,
has now been constructed, in which line the expression of the E2 or
E4 regions is directed by a promoter equipped at its 5' and with
so-called "operator" sequences of the bacterial tetracycline
operon, these sequences hereinafter being designated tet O. The
synthesis of the corresponding expression products will be
activated only in the presence of an inducer which can be produced
by the adenoviral vector or by the line itself. Similarly, a
repressor may be added to the culture medium when complementation
is no longer desired.
[0010] On the other hand, new adenoviral vectors from which the
majority of the E1 and E3 regions have been deleted have now been
generated, in which vectors the transcription units of the
remaining viral regions (E2, E4 and/or L1-L5) are regulable with
the object of permitting their expression when infectious viral
particles are to be generated and of inhibiting it in the host
cell. in the examples which follow, the regulation is effected by
the tet O sequences. Their insertion On the 5' side of the TATA box
generates a promoter from which the baseline level of transcription
in minimal but may be strongly stimulated in the presence of the
inducer mentioned above. Thus, the production of viral proteins is
activated in a 293 line expressing the inducer, which will enable
infectious viral particles to be formed. In contrast, it in
considerably reduced in the infected host cell which does not
naturally produce the inducer of bacterial origin. The regulation
of the viral genes has no effect on the expression of the exogenous
nucleotide sequence placed under the control of a promoter that
does not respond to tetracycline.
[0011] The adenoviral vectors of the present invention provide an
advantageous approach to the drawbacks inherent. in the use of the
vectors of the prior art, since they combine safety of use and ease
of production. On the one hand they may be propagated in a
conventional complementation line with a high titer compatible with
industrial requirements, and on the other hand they enable an
exogenous nucleotide sequence to be transferred in vivo, and to be
expressed stably while limiting the adverse effects (inflammatory
responses in the host). They are most especially suitable for human
gene therapy.
[0012] Accordingly, the subject of the present invention in a viral
vector, characterized in that it comprises an expression unit
containing one or more viral genes; said expression unit being
functional in a complementation cell and nonfunctional in a host
cell.
[0013] For the purposes of the present invention, a "viral vector"
is obtained from a parent virus whose genome has been modified.
These modifications may be diverse (deletion, mutation and/or
addition of one or more nucleotides) and localized in the coding
regions of the viral genome or outside these regions, for example
in the promoter regions. As a guide, some viral sequences may be
deleted, rendered nonfunctional or replaced by other sequences, and
In particular an exogenous nucleotide sequence whose expression is
sought in a host cell.
[0014] A viral vector according to the invention may be derived
from a wide variety of viruses, such as herpesviruses,
cytomegaloviruses, AAV (adeno-associated virus) and poxviruses, and
in particular vaccinia, fowlpox or canarypox virus. Such vectors,
as well as the techniques for preparing them, are known to a person
skilled in the art.
[0015] However, a vector which is especially suitable for the
present invention is an adenoviral vector. It may be derived from
an adenovirus of human, canine, avian, bovine, =urine, ovine,
porcine or simian origin, or alternatively from a hybrid comprising
adenoviral genome fragments of different origins. The adenoviruses
CAV-1 or CAV-2 of canine origin, DAV of avian origin or
alternatively Bad type 3 of bovine origin (Zakharchuk et al., 1993,
Arch. Virol., 128, 171-176; Spibey and Cavanagh, 1989, J. Gen.
Virol., 70, 165-172; Jouvenne et al., 1987, Gene, 60, 21-28; Mittal
et al., 1995, J. Gen. Virol., 76, 93-102) may be mentioned more
especially. However, an adenoviral vector derived from a human
adenovirus, preferably of serotype C and, as an absolute
preference, of type 2 or 5 (Graham and Prevect, 1991, Methods in
Molecular Biology, vol. 7, p 109-128; Ed: E. J. Murey, The Human
Press Inc.), will be preferred.
[0016] An advantageous embodiment of the present invention consists
of a vector which is defective for replication, in which one or
more viral genes necessary for replication are deleted or rendered
nonfunctional. Such a vector, incapable of autonomous replication,
will be propagated in a complementation cell. The term
"complementation cell" denotes a cell capable of supplying in trans
the function(s) for which a vector according to the invention is
defective. In other words, it is capable of producing the
proteins(s) necessary for the replication and encapsidation of said
vector, early and/or late proteins, which it cannot itself produce
and which are necessary for the formation of an infectious viral
particle. By way of illustration, since a preferred adenoviral
vector according to the invention lacks most of the E1 region, use
will be made of a complementation cell such as line 293. capable of
supplying in trans the collective proteins encoded by the E1 region
which the vector cannot produce. "Infectious viral particle" is
understood to mean a viral particle having the capacity to infect a
host cell and to cause the viral genome to enter the latter.
[0017] According to a preferential embodiment, a viral vector
according to the invention in recombinant. Thus, it will comprise
an exogenous nucleotide sequence placed under the control of the
elements necessary for its expression in a host cell. "Exogenous
nucleotide sequence" refers to a nucleic acid which can be of any
origin and which is not normally present in the genome of a parent
virus employed in the present invention or, if it is present, in a
different genomic context. In the context of the invention, the
exogenous nucleotide sequence perhaps [sic] made up of one or more
genes, and especially gene(s) of therapeutic interest.
[0018] Generally speaking, the exogenous nucleotide sequence can
code for an antisense RNA and/or an mRNA which will then be
translated into a protein of interest. It may be of the genomic
type, of the complementary DNA (cDNA) type or of mixed type
(minigene, in which at least one intron is deleted). It may code
for a mature protein or a precursor of a mature protein, in
particular a precursor intended to be secreted and comprising a
signal peptide. Moreover, the encoded product may be all or part of
a protein as found in nature (native or truncated protein), or
alternatively a chimeric protein originating from the fusion of
sequences of diverse origin or else a mutated protein displaying
improved and/or modified biological properties. Such proteins may
be obtained by the conventional techniques of molecular
biology.
[0019] In the context of the present invention, it can be
advantageous to use the genes coding for the following
polypeptides:
[0020] cytokines or lymphokines (interferons .alpha., .beta. and
.gamma., interlukins and in particular IL-2, IL-6, IL-10 or IL-12,
tumor necrosis factors (TNF), colony stimulating factors (GM-CSF,
C-CSF, M-CSF, etc.);
[0021] cell or nuclear receptors (receptors recognized by
pathogenic organisms (viruses, bacteria or parasites), and
preferably by the HIV virus (human immunodeficiency virus) [lacuna]
or their ligands;
[0022] proteins involved in a genetic disorder (factor VII, factor
VIII, factor IX, dystrophin or mini-dystrophin, insulin, CFTR
(cystic fibrosis trans-membrane conductance regulator) protein,
growth hormones (HGF) [lacuna];
[0023] anzymes (urease, renin, thrombin, etc..)
[0024] enzyme inhibitors (.alpha.1-antitrypsin, antithrombin III,
inhibitors of viral proteases, etc.);
[0025] polypeptides having an antitumor effect capable of at least
partially inhibiting the initiation or progression of tumors or
cancers (antisense RNAs, antibodies, inhibitors acting on cell
division or on transduction signals, expression products of tumor
suppressing genes, for example p53 or Rb, proteins that stimulate
the immune system, etc.);
[0026] proteins of the major histocompatibility complex classes I
or II, or regulatory proteins that act on the expression of the
corresponding genes;
[0027] polypeptides capable of inhibiting a viral, bacterial or
parasitic infection and/or its development (antigenic polypeptides
having immunogenic properties, antigenic epitopes, antibodies,
trans-dominant variants capable of inhibiting the action of a
native protein by competition, etc.);
[0028] toxins (herpes simplex virus 1 thymidine kinase (HSV-1 TK),
ricin, cholera or diphtheria toxin, etc.) or immunotoxins; and
[0029] markers (.beta.-galactosidase, luciferase, etc.).
[0030] It should be pointed out that this list is not limiting and
that other genes may also be employed.
[0031] Moreover, an exogenous nucleotide sequence employed in the
present invention may comprise, in addition, a selectable gone
enabling the transfected cells to be selected or identified. There
may be mentioned the neo gene (coding for neomycin
phosphotransferase) conferring resistance to the antibiotic G418,
the dhfr (dihydrofolate reductase) gene, the CAT (chloramphenicol
acetyltransferase) gene, the pac (purosycin acetyltransferase) gene
or alternatively the gpt (xanthine guanine
phosphoribosyltransferase) gene. Generally speaking, the selectable
genes are known to a person skilled in the art.
[0032] Elements necessary for the expression of an exogenous
nucleotide sequence in a host cell are understood to mean the
collective elements permitting its transcription into RNA
(antisense RNA or mRNA) and the translation of an mRNA into
protein. Among these, the promoter assumes special importance. It
may be isolated from any gone of eucaryotic or even viral origin,
and may be constitutive or regulable. Alternatively, it can be the
natural promoter of the gene in question. It will be preferable to
employ a promoter different from the one included in the unit for
the expression of the viral genes (defined below). Moreover, it may
be modified so as to improve the promoter activity, to abolish a
region that inhibits transcription, to render a constitutive
promoter regulable or vice versa, to introduce a restriction site,
etc. There may be mentioned, by way of examples, the promoters of
the HSV-1 TK, murine or human PGK (phosphoglycerate kinase),
.alpha.1-antitrypsin (liver-specific) and immunoglobulin
(lymphocyte-specific) genes, the SV40 virus (simian virus 40) early
promoter, a retroviral LTR or alternatively the adenoviral MLP.
promoter, in particular of hum adenovirus type 2.
[0033] Naturally, an exogenous nucleotide sequence employ d in the
present invention can, in addition, comprise further elements
necessary for expression (intron sequence, signal sequence, nuclear
localization sequence, transcription termination sequence,
translation initiation site of the IRES or an other type, etc.) or
alternatively for its maintenance in the host cell. Such elements
are known to a person skilled in the art.
[0034] As stated above, a viral vector according to the present
invention comprises an expression unit containing one or more viral
genes and having the advantageous feature of being functional in a
complementation cell and nonfunctional in a host cell. "Functional"
is understood to mean an expression of the viral genes in a
sufficient amount and for a sufficiently long time to permit the
formation of an infectious viral particle. "Nonfunctional" is
understood to moan an expression of the viral genes which is
reduced (preferably by a factor of at least 10, or even a zero
expression) relative to their level of expression in the parent
virus. The functional character manifests itself in the production
of the products of the viral genes included in the expression unit,
it being possible for these products to be demonstrated by the
standard techniques of molecular biology, immunology, biochemistry
or enzymology. The nonfunctional character manifests itself in the
absence of production or alternatively in the production of the
viral products at a reduced level.
[0035] Advantageously, said expression unit comprises one or more
heterologous regulatory sequence(s) not present in the parent viral
DNA. Use may be made of sequences that respond to a regulator of
the repressor type acting negatively on the expression, or
preferably to a regulator of the inducer type exerting a positive
action. A regulatory sequence employed in the context of the
present invention may be of any origin, viral, prokaryotic or
alternatively eukaryotic. Generally speaking, regulatory sequences
are described in the literature available to a person skilled in
the art. It is also possible to employ a homolog whose sequence is
modified relative to the native sequence but which exerts a similar
or improved regulatory function. These modifications may result
from the addition, deletion and/or replacement of one or more
nucleotides.
[0036] In accordance with the objectives pursued by the present
invention, a regulatory sequence is capable of modulating the
expression of the viral genes at different levels: transcription,
elongation, transport, stability of the mRNAs or alternatively
translation. It may be present at various places in said expression
unit, for example in the promoter, especially when its effect lies
at transcriptional level (preferably upstream of the TATA box, up
to a few hundred base pairs from the latter), or in the transcribed
(and if possible noncoding) sequences when its action in exerted at
a subsequent step of the transcription. It is possible to employ
from 1 to 25 regulatory sequences, advantageously from 1 to 20,
preferably from 1 to 10 and, as an absolute preference, from 1 to
7.
[0037] For the purposes of the present invention, the term
"inducer" denotes a molecule which has the capacity of initiating
or activating the expression of the viral genes placed under the
control of a regulatory sequence, either directly by binding to
said regulatory sequence, or indirectly via other cellular or viral
factors. It can also prevent the action of a repressor. In
contrast, a "repressor" has the capacity to inhibit or block the
expression of the viral genes placed under the control of a
regulatory sequence on which it acts, this taking place either
directly or indirectly.
[0038] These definitions may be illustrated by the example of the
lactose (lac) operon. The lac gone codes for a repressor which
binds to a short regulatory sequence termed "operator", thereby
preventing the transcription of the structural genes coding for the
enzymes of the metabolic pathway of lactose. In the presence of the
inducer, the latter binds to the repressor and converts it to an
inactive form which can no longer bind to the operator, thereby
enabling transcription to take place.
[0039] It is also possible to nvisage using portions or analogs of
these regulators in order to improve their efficacy or modify.
their specificity (for example anhydrotetracycline, ten times as
effective as tetracycline for inhibiting transcription from a
promoter comprising the regulatory sequences derived from the
tetracycline operon, or the reverse transactivator described
recently by Gossen at al., 1995, Science, 268, 1766-1769).
Moreover, a regulator employed in the present invention can be a
hybrid protein originating from the fusion of polypeptides of
different origins. A preferred combination consists of a
polypeptide capable of recognizing or binding a regulatory sequence
employed in the present invention (for example derived from the
tetracycline repressor (tet R) or estrogen repressor ER) and a
polypeptide capable of activating expression (for example the
activation domain of the Ga14 or VP16 proteins, capable of
interacting with transcription factors).
[0040] Table 1 below lists some of the regulatory sequences and
regulators which can be used in the context of the present
invention:
1 Inducer (+)/ Origin of the regulatory sequences Repressor (-)
Insertion site Bibliographic reference MRE (metal-responsive
element) metal ions (+) 5' TATA Makarov et al., 1994,
metallothionein gene Nucleic Acids Res. 22, 1504-1505 tryptophan
operon tryptophan (-) 5' TATA Yanofsky et al., 1981, Nucleic Acids
Res. 9, 6647-6668 lac operator product of the lacI gene (-) 5' TATA
Miller and Reznikoff (Eds), The operons (Cold Spring Harbor
Laboratory, New York (lacuna) tet operator VP16-TetR protein (+) 5'
TATA Gossen and Bujard, 1992, Proc. Natl. Acad. Sci. USA 89,
5547-5551 tet operator TetR (-) 3' TATA Kim, 1995, J. Virol. 69,
2565-2573 TAR (transactivation responsive TAT (+) transcribed
Steffy and Wong-Staal, region) 3' sequence 1991, Microbiological
Reviews 55, 193-205 RRE (REV responsive element) REV (+)
transcribed Steffy and Wong-Staal, 3' sequence 1991,
Microbiological Reviews 55, 193-205 GRE (glucocorticoid responsive
glucocorticoid (+) 5' TATA Israel and Kaufman, 1989, element)
Nucleic Acids Res. 17, 4589-4604 PRE (progesterone responsive
progesterone (+) 5' TATA Gronemeyer et al., 1987, element) EMBO J.
6, 3985-3994 Ga14 UAS (Ga1 4 upstream Ga14 (+) 5' TATA Webster et
al., 1988, Cell activating sequence) 52, 169-178 ERE (estrogen
responsive element) Estrogen (+) 5' TATA Klein-Hitpa.beta. et al.,
1986, Cell 46, 1053-1061 The insertion site is given only as a
guide but without implied limitation
[0041] According to a particular embodiment of the present
invention, regulatory sequences derived from the bacterial
tetracycline operon, designated in the literature "operator" (tet
O), are used. Generally speaking, the tetracycline resistance
operon in encoded by the transposon TN10 (Hillen et al., 1984, J.
Mol. Biol. 172, 185-201). Regulation is effcted by a short
nucleotide sequence termed "operator" (tet O) which constitutes a
binding site for various regulators. Thus, the binding of the
tetacycline [sic] repressor (tet R) or of the antibiotic
tetracycline considerably decrease the level of transcription. On
the contrary, an activation effect is obtained by employing a
protein, designated in the literature "tetracycline transactivator
(tTA)", which results from the fusion between tat R and the. 130
C-terminal a acids of the activation domain of the VP16 protein of
the herpes simplex virus. Goosen and Boujard (1992, Proc. Natl.
Acad. Sci. USA 89, 5547-5551) have recently shown that this
regulatory system is functional in eukaryotic cells. The expression
of a reporter gone placed under the control of several copies of
tet O upstream of basic transcription sequences (TATA box,
transcription startsite, etc.) in detectable by coexpression of tTA
and inhibited by the addition of tetracycline. Rim's group (1995,
J. Virol. 69, 2565-2573), for its part, utilizes the tet O
sequences positioned downstream of the TATA box of a promoter, and
in this case transcription is inhibited by the action of tetR.
[0042] In the context of the present invention, the combination
"tet O-minimal promoter" (in the 5' to 3' direction), giving rise
to a promoter whose baseline transcription level in naturally very
low but activable by the inducer tTA and repressible by
tetracycline, is most especially preferred. However, it in also
possible to use a promoter in which the tet O sequences are placed
on the 3' side of the TATA box, or alternatively on each side of
the latter, and which is repressible by a repressor comprising the
sequences of tet R that recognize tet O.
[0043] As regards the preferred variant, an adenaoviral vector
according to the invention preferably consists of the genome of an
adenovirus lacking all or part of the E1 region and, alternatively,
all or part of the E3 region. Advantageously, it in preferable to
retain a portion of the E3 region, and in particular the portion
corresponding to the gene coding for the gp19k (Gooding and Wold,
1990, Critical Reviews of Immunology 10, 53-71), said portion not
being included in an expression unit as defined above but placed
under the control of a conventional homologous (E3) or heterologous
promoter. It is self-evident that it is possible to carry out other
modifications of the viral genome, in particular in the E4 or E2
region. It may be advantageous to introduce mutations or additional
deletions. To illustrate this point, the temperature-sensitive
mutation affecting the DBP (standing for DNA binding protein) gene
of the E2A region (Ensinger and Ginsberg, 1972, J. Virol. 10,
328-339).
[0044] According to a preferential embodiment, an adenoviral vector
according to the invention comprises an expression unit containing
one or more viral genes of the E2, E4 or L1-L5 regions. An
advantageous variant consists in retaining from the E4 region only
the sequences coding for ORFs 3, 6 and/or 7 (these limited
deletions of the E4 region not necessitating complementation of the
E4 function; Ketner et al., 1989, Nucleic Acids Res. 17,
3037-3048).
[0045] It can also be advantageous to have at one's disposal an
adenoviral vector comprising several expression units, it being
possible to envisage all the combinations (E2 and E4, E2 and L1-L5,
E4 and L1-L5 or E2, E4 and L1-L5). Regulatory sequences acting in
the same manner, and preferably positively (for example TAR, RRE,
tet O, etc.), will then be chosen. For reasons of simplicity of
implementation, preference is given to the case where the units
carry identical regulatory sequences enabling the adenoviral vector
to be propagated in a complementation line comprising a single
inducer.
[0046] The invention also relates to an infectious viral particle,
as well as to a eukaryotic host cell comprising a viral vector
according to the invention. Said host cell is advantageously a
mammalian cell, and preferably a human cell, and can comprise said
vector in integrated form in the genome or in nonintegrated form
(episome). It can be a primary or tumor cell of hematopoietic
(totipotent stem cell, leukocyte, lymphocyte, monocyte or
macrophage, etc.), muscular, pulmonary, hepatic, epithelial or
fibroblast origin.
[0047] The subject of the present invention is also a
complementation cell, characterized in that it comprises an inducer
and/or a repressor (regulator). Depending on the requirements, the
latter may be added to the culture medium, or produced stably or
transiently by the cell itself. Preferably, a complementation cell
according to the invention is modified by the introduction of a DNA
fragment coding for said regulator. All standard means for
introducing a nucleic acid (synthetic, viral or plasmid vector,
naked DNA, etc.) into a cell may be used in the context of the
present invention, such as, for example, transfection,
electroporation, microinjection, lipofection, adsorption and
protoplast fusion. In the context of the invention, it can be
advantageous to generate a complementation cell which produces only
a single regulator, and in particular an inducer. However, it can
also be advantageous to have at one's disposal cells producing
several inducers.
[0048] According to an advantageous embodiment, a complementation
cell according to the invention is capable of complementing In
trans a viral vector according to the invention, and especially an
adenoviral vector. In this connection, a complementation cell for
the E1 and/or E4 function will advantageously be chosen.
Accordingly, the invention also relates to a cell intended for the
complementation of a second generation adenoviral vector which is
defective for the E1 function and another adenoviral function (late
or early). Such a cell comprises all or part of the 3E region of an
adenovirus whose expression is controlled by any promoter, and all
or part of a region of an adenovirus other than the E1 region,
placed under the control of a promoter equipped with regulatory
sequences, for example at its 5' end with at least one and
preferably one to 20 tet O sequence(s) which is/are activable by
the transactivator tTA. An advantageous variant consists of a
minimal promoter derived from the CMV virus (cytomegalovirus),
upstream of which lie 7 tat O sequences in a head-to-tail
orientation. The inducer may be introduced into the complementation
cell according to the invention prior to, concomitantly with or
subsequently to the adenoviral sequences placed under the control
of the tet O sequences or, as mentioned above, it may be added to
the culture medium. It in also possible to envisage expressing the
inducer (for example tTA) in the complementation cell, and adding
the repressor (for example tetracycline) to the culture medium when
the expression of the adenoviral genes is no longer desired.
[0049] By way of preferred examples, the adenoviral region other
the E1 region consists of:
[0050] (i) all or part of the E4 region, and in particular the
sequences coding for open reading frames 6 and 7 (ORFs 6/7) of the
latter, or alternatively
[0051] (ii) all or part of the E2 region, and in particular the
sequences coding for the DBP protein (DNA binding protein) or a
temperature-sensitive mutant of the latter.
[0052] Naturally, a complementation cell according to the invention
can, in addition, comprise a third adeno-viral region whose
expression is placed under the control of the appropriate elements.
In this connection, a preferred cell is intended for the
complementation of an adenoviral vector which is defective for the
collective early functions which are essential for replication, and
comprises all or part of. the E1, E2 and E4 regions, one or other
or both of the latter two regions being placed under the control of
a promoter equipped with regulatory sequence(s) as defined
above.
[0053] One of the advantages of a complementation cell according to
the invention is that it permits the production at high titer of
infectious viral particles from a conventional viral vector or
viral vector according to the invention. The viral titer of the
final preparation (after purification) is advantageously greater
than 5.times.10.sup.9 pfu/ml, preferably greater than
1.times.10.sup.9 pfu/ml, as an absolute preference greater than
5.times.10.sup.9 pfu/ml and, as yet a further preference, greater
than 5.times.10.sup.10 pfu/ml. The term pfu is understood to refer
to a particle capable of forming a plaque by infection of
permissive cells. The techniques for evaluating the number of pfu
are conventional and known to a person skilled in the art. The agar
technique (Graham and Prevec, 1991, supra) may be mentioned.
Generally, the viral titer in pfu/ml is equal to or less than the
actual number of infectious viral particles capable of carrying out
their gene transfer function. In effect, a certain percentage is
incapable of propagating effectively and hence of generating lytic
plaques on permissive cells. The titer of infectious viral
particles produced by a complementation cell according to the
invention is advantageously greater than 5.times.10.sup.9 ifu/ml,
preferably greater than 1.times.10.sup.10 ifu/ml, as an absolute
preference greater than 5.times.10.sup.10 ifu/m [sic] and, as yet a
further preference, greater than 5.times.10.sup.11 ifu/ml. The term
ifu is understood to refer to an infectious particle capable of
infecting a nonpermissive target cell and of transferring its
genome and permitting the expression of the genes carried by the
latter. The number of ifu in estimated by the number of target
cells expressing the gone of interest or a viral gone. A person
skilled in the art is aware of the techniques to be employed for
detecting their expression: by immunofluorescence, western blotting
or alternatively staining, etc. For example, when the gene of
interest consists of the LacZ gone, the protein
.beta.-galactosidase may be visualized by staining with X-Ga1
(5-bromo-4-chloro-3-indolyl .beta.-D-galactopyranoside) and the
number of blue cells is counted. When the CFTR therapeutic gone is
employed, the expression product may be visualized by western
blotting. It in also possible to look for cells expressing the
adenoviral DBP, penton or fiber proteins using specific
antibodies.
[0054] A complementation-cell according to the invention may be
generated from various cell lines by transfection of suitable
portions of the adenoviral genome and a DNA fragment coding for a
regulator. Among the lines which can be envisaged, there may be
mentioned the Vero kidney (monkey), BHK (hamster), MDCK (dog) and
MBDK (bovine) lines, the CHO line (hamster) or alternatively the
human lines (HeLa, A549, MRC5, W138, etc.) available in collections
such as the ATCC (Rockville, USA). However, an especially suitable
cell is line 293. The use of primary lines such as primary human
retina cells may also be envisaged.
[0055] An infectious viral particle according to the invention may
be prepared according to any conventional technique in the field of
the art (Graham and Prevect, 1991, supra), for example by
cotransfection of a vector and an adenoviral fragment into a
suitable cell, or alternatively by means of a helper virus
supplying in trans the nonfunctional viral functions. It is also
possible to envisage generating the viral vector in vitro in
Escherichia coli (E. coli) by ligation or alternatively homologous
recombination (see, for example, French Application 94/14470).
[0056] The invention also relates to a method of preparation of an
infectious viral particle comprising a viral vector according to
the invention, according. to which:
[0057] (i) said viral vector in introduced into a complementation
cell capable of complementing in trans said vector, so as to obtain
a transfected complementation cell,
[0058] (ii) said transfected complementation cell in cultured under
suitable conditions to permit the expression of th viral genes and
the production of said infectious viral particle, and
[0059] (iii) said infectious viral particle is recovered in the
cell culture.
[0060] Naturally, the infectious viral particle may be recovered
from the culture supernatant, but also from the cells. According to
an advantageous embodiment, an adenoviral vector and a
complementation cell according to the invention are employed.
According to another variant, use may be made of a conventional
complementation cell. It may be necessary to add an inducer to the
culture medium in the case where the expression unit comprises
activable regulatory sequences. The amount to be used depends on
the actual nature of the inducer. A person skilled in the art will
quite obviously be able to Adapt the optimal concentration in
accordance with the specific data.
[0061] The invention also relates to a method of preparation of an
infectious viral particle comprising a conventional viral vector,
employing a complementation cell according to the invention. By way
of example, the introduction of a viral vector which is defective
for the E1 and E4 functions (E1- E4-) into a 293 cell expressing
(i) ORFs 6/7 of the E4 region under the control of a minimal
promoter equipped at its 5' and with 7 tet O sequences, and (ii)
the. transactivator tTA directed by the same promoter, will ale
viral particles to be generated which are deficient for replication
but infectious with respect to a host cell.
[0062] The subject of the invention in also a pharmaceutical
composition comprising as therapeutic or prophylactic agent a viral
vector, an infectious viral particle, a complementation cell or a
eukaryotic host call according to the invention, in combination
with a vehicle which is acceptable from a pharmaceutical stand-
point. The composition according to the invention in intended
especially for the preventive or curative treatment of disorders
such an:
[0063] genetic disorders (hemophilia, cystic fibrosis, diabetes or
myopathy, Duchne's [sic] myopathy and Becker's myopathy, etc.),
[0064] cancers, such as those induced by oncogenes or viruses,
[0065] viral diseases such as hepatitis B or C and AIDS (acquired
immunodeficiency syndrome resulting from infection with HIV),
and
[0066] recurrent viral diseases such as the viral infections caused
by herpesvirus.
[0067] A pharmaceutical composition according to the invention may
be manufactured in a conventional manner. In particular, a
therapeutically effective amount of a therapeutic or prophylactic
agent in combined with a vehicle such as a diluent. A composition
according to the invention may be administered locally,
systemically or by aerosol, especially via the intragastric,
subcutaneous, intracardiac, intramuscular, intravenous,
intraperitoneal, intratumoral, intrapulmonary, intranasal or
endotracheal route. The administration may take place in a single
dose or a dose repeated one or several times after a certain time
interval. The appropriate administration route and dosage vary in
accordance with various parameters, for example with the individual
or the disorder to be treated or alternatively with the gene(s) of
interest to be transferred. In particular, the viral particles
according to the invention may be formulated in the form of doses
of between 10.sup.4 and 10.sup.14 pfu (plaque forming units),
advantageously 10.sup.5 and 10.sup.13 pfu and preferably 10.sup.6
and 10.sup.11 pfu. The formulation can also include an adjuvant or
an excipient which is acceptable from a pharmaceutical
standpoint.
[0068] Lastly, the present invention relates to the therapeutic or
prophylactic use of a viral vector, of an infectious viral
particle, of a complementation cell or of a eukaryotic host cell
according to the invention, for the preparation of a medicinal
product intended for the treatment of the human or animal body, and
preferably by gene therapy. According to a first possibility, the
medicinal product may be administered directly in vivo (for example
by intravenous injection, in an accessible tumor, in the lungs by
aerosol, etc.) . It is also possible to adopt the ex vivo approach,
which consists in removing cells from the patient (bone marrow stem
cells, peripheral blood lymphocytes, muscle calls, etc.),
transfecting or infecting them in vitro according to the techniques
of the art and readministering then to the patient. In the case
where the expression unit comprises regulator sequences that
respond to a repressor, it is possible to envisage administering
the repressor in order to block or limit the expression of the
viral genes (prior, concomitant or subsequent administration of the
repressor or coexpression via conventional vectors).
[0069] The invention also extends to a method of treatment,
according to which a therapeutically effective amount of a viral
vector, of a viral particle, of a eukaryotic host cell or of a
complementation cell according to the invention is administered to
a patient requiring such treatment.
[0070] The present invention is described more fully by reference
to the figures which follow and by means of the examples which
follow.
[0071] FIG. 1 is a diagrammatic representation of the genome of the
human adenovirus type 5 (represented in arbitrary units from 0 to
100), showing the location of the different genes.
[0072] FIG. 2 is a diagrammatic representation of the vector
pTG4673, permitting the constitutive expression of tTA directed by
the CMV promoter (pCMV) and of the pac (puromycin resistance)
selectable gone under the control of the SV40 promoter.
[0073] FIG. 3 is a diagrammatic representation of the adenoviral
vector pTG4696 containing a cassette for the expression of ORFs 6
and 7 of the E4 region, activable by tTA.
[0074] FIG. 4 is a diagrammatic representation of the vector
pTG8343 containing a portion of the 5' end of the adenovirus 5
genome.
[0075] FIG. 5 is a diagrammatic representation of the vector
pTG4662, a first generation recombinant adenoviral vector from
which the bulk of the E1 and E3 regions has been deleted. The
recombinant cassette consists of the adenoviral MLP promoter (Ad2),
followed by the bacterial LacZ gone coding for .beta.-galactosidase
and the SV40 virus polyadenylation signal (pA).
[0076] FIG. 6 in a diagrammatic representation of the vector
pTG4682, an adenoviral vector from which the bulk of the E1 and E3
regions has been deleted and containing a cassette for the
constitutive expression of tTA.
[0077] FIG. 7 in a diagrammatic representation of the vector
pTG4683, an adenoviral vector from which the bulk of the E1, E3 and
E4 regions has been deleted and containing a cassette for the
constitutive expression of tTA.
[0078] FIG. 8 is a diagrammatic representation of the vector
pTG4675, permitting the inducible expression of tTA (under the
control of the CMV minimal promoter (-53 to +1) preceded by 7
copies of tet O sequences, indicated on the figure by tet
O-CMVi).
[0079] FIG. 9 is a diagrammatic representation of the vector
pTG4684, an adenoviral vector from which the bulk of the E1 and E3
regions has been deleted and containing a cassette for the
inducible expression of tTA.
[0080] FIG. 10 is a diagrammatic representation of the vector
pTG4685, an adenoviral vector from which the bulk of the E1, E3 and
E4 regions has been deleted and containing a cassette for the
inducible expression of tTA.
[0081] FIG. 11 is a diagrammatic representation of the vector
pTG5606, permitting the expression of ORFs 6/7 of the adenoviral E4
region and of tTA under the control of the CMV minimal promoter
preceded by 7 copies of tet O sequences and the pac selectable gone
conferring puromycin resistance.
[0082] FIG. 12 is a western blot analysis of the expression of the
DBP protein in 293 cells transfected transiently with the vector
pTG9579 and cultured in the presence (+) and in the absence (-) of
tetracycline.
EXAMPLES
[0083] The examples which follow illustrate just one embodiment of
the present invention.
[0084] The constructions described below are carried out according
to the general techniques of genetic engineering and molecular
cloning detailed in Maniatis et al., (1989, Laboratory Manual, Cold
Spring Harbor, Laboratory Press, Cold Spring Harbor, N.Y.) or
according to the manufacture's recommendations when a commercial
kit is used. Cloning steps employing bacterial plasmids are carried
out in E. coli strain 5K (Hubacek and Glover, 1970, J. Mol. Biol.
50, 111-127) or BJ 5183 (Hanahan, 1983, J. Mol. Biol. 166,
557-580). The latter strain in preferentially used for the
homologous recombination steps. The PCR amplification techniques
are know to a person skilled in the art (see, for example, PCR
Protocols-A guide to methods and applications, 1990, edited by
Innis, Gelfand, Sninksy and White, Academic Press Inc). As regards
the repair of restriction sites, the technique employed consists of
filling in the 5' protruding ends using the large fragment of E.
coli DNA polyserase I (Klenow).
[0085] As regards the cell biology, the cells are transfected
according to the standard techniques well known to a person skilled
in the art. The calcium phosphate technique (Maniatis at al.,
supra) may be mentioned, but any other protocol may also be
employed, such as the DEAE-dextran technique, electroporation,
methods based on osmotic shock, microinjection or methods based on
the us* of liposomes. The culture conditions are, for their part,
conventional.
[0086] In the examples which follow, the following cell lines are
employed:
[0087] Line 293 derived from human embryonic kidney (Graham et al.,
1977, supra), which results from the integration in its chromosomes
of the 5' and of the Ad5 genome (5'ITR, encapsidation sequence and
E1 region) (available at the ATCC under the reference CRL
1573).
[0088] Line TG1653 (described in International Application
WO94/28152, Example 8), which in derived from line 293 stably
transformed by the plasmid pTG1653 carrying the Ad5 E4 region (nt
32800 to 35826) and the cassette for the expression of the pac
selectable gone.
[0089] It should be understood that other cell lines may be
used.
[0090] Moreover, the adenoviral genome fragments employed in the
different constructions described below are indicated precisely
according to their position in the nucleotide sequence of the Ad5
genome as disclosed in the Gen databank under the reference
M73260.
EXAMPLE 1
[0091] Complementation Line Expressing an Inducer Capable of
Activating the Expression of Adenoviral Genes
[0092] This example describes the construction of (1) a
complementation line derived from line 293 and capable of
expressing constitutively the transactivating protein tTA, and (2)
an adenoviral vector in which some viral genes of the E4 region
have boon placed under the control of tet O sequences responding to
tTa. The transfection of the vector into the line is followed by
the formation of viral particles, since the E1 and E4 functions are
transcomplemented by the supply of the expression products of the
adenoviral E1 region and the tTa. In infected host cells which do
not naturally produce the chimeric tTA protein, the expression of
B4 and consequently of the late proteins (E4-dependent expression)
will be considerably reduced, thereby limiting the problem of
cellular immunity and inflammation.
[0093] 1. Construction of a Complementation Line Expressing the
Transactivator tTA
[0094] The vector pTG6529, permitting the selection of the
transformed cells with puromycin, is constructed first. The vector
results from the cloning into a p polyIII-I.sup.+(Lathe et al.,
1987, Gone 57, 193-201) of an expression cassette composed of the
SV40 promoter and the pac gone followed by the SV40 virus polyA
signal. Such a construction is within the capacity of a person
skilled in the art, from the corresponding sequences described in
the literature (Morgenstern and Land, 1990, Nucleic Acid Res. 18,
3587-3596; Genebank reference JO2400).
[0095] In parallel, the vector pUHD15-1 (Gossen and Bujard, 1992,
supra) is digested with the enzymes SspI and HpaI. The fragment
containing the sequences coding for tTa preceded by the CMV
promoter is cloned between the same sites of pTG6529 to give
pTG4673 (FIG. 2).
[0096] The vector pTG4673 is transfected in a conventional manner
into 293 cells. Naturally, it would also have been possible to
transfected a vector for the expression of tTA and a selectable
vector (for example puHD15-1 and pTG6529). After transfection, the
cells are cultured in selective medium (1 .mu.g/ml of puromycin).
80 resistant clones were isolated and, among these, 35 tested for
their capacity to transactivate a marker gone whose expression is
controlled by the tet O sequences. For reasons of simplicity of
implementation and of sensitivity of assay, the choice fell on the
luciferase gone.
[0097] The reporter vector pUHC13-3 (Gossen and Bujard, 1992,
supra) in used, in which the sequences coding for luciferase are
placed under the control of the CMV minimal promoter preceded by 7
copies of tet O. The test in performed by transient transfection
into the clones to be tested. Two days later, the luciferase
activity is evaluated on the cell extracts employing a kit marketed
by Promega ("Luciferase Assay System") and, for the measurement of
the samples, a scintillation counter (LS 50000 TD, Beckman). The
assay proves positive for 10 of the clones evaluated, indicating
that they produce a functional tTA product capable of recognizing
the tet O sequences and of activating the expression of the genes
placed-under their control.
[0098] 2. Construction of an Adenovirus in which the Expression of
the E4 Genes Is Inducible with tTa.
[0099] As mentioned above, the expression of the genes coding for
ORFs 6 and 7 of the E4 region in sufficient to effect viral
replication without the need for complementation. This example
describes the construction of an adenoviral vector from which the
E1 and E3 regions and a portion of E4 have been deleted, with the
exception of the sequences coding for ORFs 6 and 7 whose expression
is placed under the control of tet O and, as a result, stimulated
in the presence of tTA.
[0100] The vector pUHD10-3 originates from the cloning into plasmid
pBR322 of an XhoI-EcoRI fragment carrying 7 tet O copies and the
CMV minimal promoter (position -53 relative to the transcription
startsite; see Gossen and Bujard, 1992, supra). After digestion
with BamHI (site located downstream of the promoter region), the
Bg1II-BamHI fragment obtained from pTG1653 and carrying the
sequences coding for ORFs 6 and 7 is inserted to give pTG4658.
[0101] The vector pTG4664 results from the cloning of the KpnI-HpaI
adenoviral fragment (nucleotides 25 838 to 32 004) between the
KpnI-SmaI sites of a p poly II (Lathe et al., 1987, supra) in which
the Bg1II site had been abolished beforehand. A large portion of E3
(nucleotides 27 871 to 30 748) in then deleted by enzymatic
digestion (HpaI -HindIII).
[0102] The XhoI-HpaI fragment carrying the cassette for the
regulated expression of ORFs 6 and 7, is isolated from pTG4658 and
inserted after the action of the Klenow fragment into the vector
pTG4664 digested with Bg1II and treated with the Klenow fragment,
to give pTG4668 and pTG4669 according to the orientation of the
cassette. The latter is introduced into an adenoviral vector by
homologous recombination. The recombinant vector pTG4663, which is
derived from a second generation vector (from which the E1 and B4
regions essential for replication and the nonessential E3 region
have been deleted (deletion of nucleotides 27 871 to 30 748);
described in Example 4 of International Application WO94/28152),
into which the "MLP promoter-LacZ gone SV40 pA" expression cassette
has been cloned in place of the E1 region, is used for this
purpose. E. coli cells are cotransformed with pTG4668 or, pTG4669
cleaved with HindIII and the viral vector pTG4663 linearized with
SpeI to generate pTG4696 (FIG. 3) and pTG4697.
[0103] Other adenoviral vectors of similar type can be generated.
It is possible, for example, to envisage placing the genes of the
E2 region under the control of the tet O sequences. A person
skilled in the art is familiar with the molecular biology
techniques which enable the E2 promoter to be replaced by a
regulable promoter such as the one isolated from the vector
pUHD10-3, or the sequences upstream of the TATA box of the E2
promoter to be removed and one or more copies (preferably 7) of tet
O sequences to be introduced in their place. It is also possible to
construct vectors regulated at several level*, for ale by insertion
of tet O upstream of the TATA box of the promoters directing the
expression of the viral genes of E2, E4 and/or MLP.
[0104] 3. Generation of Viral Particles.
[0105] The viral vector pTG4696 or pTG4697 is transfected into one
of the 10 clones producing tTA of Example 1. The cello are capable
of complementing the E1 function and of producing tTa activating
the expression of ORFs 6 and 7, which permits the formation of
viral particles. A stock is built up which then enables the target
cells to be infected at a particular m.o.i. (multiplicity of
infection) capable of varying from 0.1 to 100.
EXAMPLE 2
[0106] Construction of the Complementation Line 4677 for the E1 and
E4 Functions, the Expression of E4 Being Inducible with tTA.
[0107] This example relates to a complementation line generated by
transfection of 293 cells with a vector for the expression of ORFs
6 and 7 of E4 placed under the control of a minimal promoter
preceded by 7 copies of tet O. The transfected cells are capable of
complementing the E1 functions constitutively and the E4 functions
inducibly with tTa. The latter may be carried by a defective
adenoviral vector or a conventional expression vector.
[0108] 1. Construction of the Complementation Line.
[0109] The XhoI-BamHI fragment of pTG4658 (carrying the "tet O-CMV
minimal promoter-ORFs 6 and 7" cassette) is inserted between the
Sa1I and Bg1II sites of the selection vector pTG6529 to give
pTG4677. 60 resistant clones were generated after transfection into
line 293 and selection with puromycin. The clones displaying
optimal capacity for complementation may be determined in different
ways.
[0110] A first method consists in transfecting transiently the
plasmid pUHD15-1 constitutively expressing tTA. Western blot
analysis of cell extracts using suitable antibodies will enable the
mount of ORFs 6 and/or 7 polypeptides produced in these clones, and
hence their capacity for transcomplementing the E4 adenoviral
function, to be evaluated. According to another method, a defective
adenovirus expressing tTA may be employed. This method is detailed
below.
[0111] 2. Construction of a Defective Adenovirus Constitutively
Expressing tTA.
[0112] Two series of constructions were carried out. pTG4682
corresponding to the adenoviral genome from which the bulk of the
E1 and E3 regions has been deleted, and containing a cassette for
the constitutive expression of tTA (CMV promoter) in place of the
E1 region. [sic] In addition, the E4 region is deleted from
pTG4683.
[0113] The starting material is the vector pTG8343, which
originates from the insertion into p poly II (Lathe et al., 1987,
supra) of a portion of the 5' end of the adenoviral genome, namely
the sequences extending from nucleotides 1 to 458 and 3328 to 5788
(FIG. 4). After digestion of pTG8343 with Bg1II and treatment with
the Klenow fragment, the vector in ligated with the SspI-HpaI
fragment of pUHD15-1 carrying the CMV promoter followed by the tTA
sequences. pTG4674 in obtained, intended to permit the insertion of
the tTA cassette by homologous recombination with an adenoviral
vector comprising homologous sequences. For this purpose, the
vector pTG4662 (FIG. 5) is chosen, which contains the 5' ITR and
the encapsidation sequences of Ad5 (nt 1 to 458), a cassette for
the expression of th LacZ gene in place of the E1 region and the
remaining adenoviral sequences from the E3 region has been deleted
(nucleotides 3329 to 27870 and 30749 to 35935).
[0114] The E. coli strain BJ is cotransformed with the vectors
pTG4662 linearized with ClaI and pTG4674 cleaved with SgrAI and
BatEII to obtain pTG4682 (FIG. 6). This homologous recombination
event permits an exchange of the LacZ cassette of pTG4662 for the
tTA cassette carried by the fragment originating from pTG4674.
[0115] The vector pTG4683 (FIG. 7) in obtained according to the
same technology, by in vitro homologous recombination between
pTG4663 cleaved with ClaI and pTG4674 digested with SgrAI and
BstEII. The vector pTG4663 is similar to pTG4662, except for the
fact that the bulk of the E4 region (nucleotides 32994 to 34998)
has been deleted.
[0116] 3. Construction of a Defective Adenovirus Inducibly
Expressing tTA.
[0117] To monitor the expression of the tTA sequences, use is made
of the CMV minimal promoter containing at the 5' end 7 copies of
tet O sequences, which makes the system self-inducible. In effect,
the production of a few tTA molecules from the uninduced promoter
will enable the system to be activated. As before, two adenoviral
vectors, pTG4684 and pTG4685, from which the E4 region has or has
not been deleted, are generated.
[0118] The CMV promoter of pUHD15-1 is exchanged for its regulable
homolog isolated from pUHD10-3 in the form of a XhoI-EcoRI
fragment. pTG4659 is obtained. The cassette is isolated from the
latter by digestion with SspI and HpaI, and cloned into the vector
pTG8343 linearized with Bg1II and treated with the Klenow fragment
to give pTG4675 (FIG. 8). The E. coli strain BJ is then
co-transformed with the above vector treated with SgrAI and BstEII
and pTG4662 or pTG4663 linearized with the enzyme ClaI, so as to
generate by homologous recombination the viral vectors pTG4684
(FIG. 9) and pTG4685 (FIG. 10).
[0119] The viral particles may be obtained by simple transfection
of a suitable cell line such as that of Example 2.1 in the absence
of tetracycline (the addition of the antibiotic to the culture
medium having an inhibitory effect on the transcription of tTA and
ORFs 6 and 7, which is regulated by the tet O sequences).
[0120] 4. Functional Tests of Microinfection and of
Microtitration.
[0121] The experiments are carried out in parallel on 293 cells
complementing the E1 function and 1653 cells complementing the E1
and E4 functions. The cells are distributed in a culture plate on
the basis of approximately 5.times.10.sup.4 cells per well. They
are then transfected with the vectors to be tested, pTG4682 to
4685. Their capacity for forming plaques in both cell types
(according to their rate of appearance) is evaluated, as well as
their size. As controls, the recombinant vectors pTG-4662 and
pTG4663 which correspond, respectively, to first generation
adenoviral vectors (deletion of E1 and E3) and second generation
adenoviral vectors (additional deletion of E4), and which do not
express tTA, are used.
[0122] The generation of viral particles from the construction
pTG4662 is readily detectable in both cell lines (formation of
large plaques appearing rapidly). pTG4663, for its part, can be
propagated only in 1653 cells complementing E1 and E4, and gives
small plaques which appear late.
[0123] As regards the vectors pTG4682 and pTG4684, their
transfection into 293 and 1653 cells is rapidly followed by the
formation of large, readily identifiable plaques (behavior similar
to the pTG4662 control). The vectors pTG4683 and pTG4685 from which
the 14 region has been deleted behave comparably to pTG4663: in
line 1653 the viral particles appear slowly, forming small-sized
plaques, whereas in line 293 no plaques are detected. These data
appear to indicate that the expression of tTA in not prejudicial to
viral propagation or toxic for cell growth.
[0124] As mentioned above, this technology may be applied to the
line of Example 1 (293/pTG4673) and vectors pTG4696 and
pTG4697.
EXAMPLE 3
[0125] Construction of the Complementation Line 5606 for the E1 and
E4 Functions, in which the Expression of E4 Is Inducible with the
tTA Produced by the Line.
[0126] This example describ s a complementation cell line
permitting the propagation of E1.sup.31 and E4.sup.31 deficient
adenovirus, obtained by transfection of 293 cells with the vector
pTG5606 carrying, besides the pac selectable gene, the sequences
coding for ORFs 6/7 and for tTA placed under the control of the
promoter hereinafter designated pCMV.sup.+, corresponding to the
CMV minimal promoter (position -53 relative to the transcription
startsite) equipped at the 5' end with 7 consecutive tet O
sequences. This self-inducible system makes possible, in the first
place, the production of a few tTA molecules from the minimal CMV
promoter, which will be able to act positively on the tet O
sequences and amplify their own synthesis and that of the ORF6/7
products in a sufficient amount for an effective complementation of
the defective vectors.
[0127] 1. Construction of Plasmid pTG5606 The first step is to
construct the vector pTG4688, which results from the cloning of the
cassette (CMV promoter/enhancer-tTA), purified from pTG4673
(Example 1.1) cleaved with HpaI and PvuI, into the vector pTG4677
(Example 2.1) digested with then* same enzymes. The vector pTG5606
is obtained from the above vector by replacement of the CMV
promoter/enhancer by the regulable promoter pCMV.sup.+. To this
end, pTG4688 is linearized with the enzymes ScaI-XbaI before
inserting the ScaI-XbaI fragment carrying pCMV.sup.+ isolated from
pTG4659. Plasmid pTG5606 thus generated (FIG. 11) comprising [sic]
the following three expression cassettes:
[0128] a first cassette composed of the pCMV.sup.+ promoter, the
sequences coding for the tTA transactivator and the SV40 virus
polyA sequences,
[0129] a second cassette composed of the puromycin resistance gene
(pac gene) under the control of the early promoter and the SV40
virus pA sequences, and
[0130] a third cassette composed of the pCMV.sup.+ promoter
followed by the sequences coding for the adenovirus 5 ORFs 6/7
equipped with their own polyA signal.
[0131] 2. Generation of the Complementation Line 5606.
[0132] 4.times.10.sup.6 293 cells (ATCC CRL1573) are cultured in
DMEM medium (Dubelco's [sic] modified Eagle's medium) in the
presence of 10% FCS. At around 70 to 80% confluence, they are
distributed in several dishes and are then transfected with 10
.mu.g or 20 .mu.g of plasmid pTG5606 (Gibco-BRL transfection kit;
ref. 530-8120SA). After 48 hours, the cell. are cultured in
selective medium (puromycin 0.7 .mu.g/al for the first week and
thereafter 1 .mu.g/l). The resistant clones are amplified in the
above selective medium, where appropriate in the presence of
tetracycline. The latter exerts a repressor effect on the tet O
sequences, and the object of adding it is to reduce the synthesis
of the expression products of ORFs 6/7 which might prove cytotoxic
and lead to call death. Thus, throe batches were constituted
according to the tetracycline concentration contained in the
medium, 0 .mu.mg/ml, 1 .mu.g/ml and 5 .mu.g/al, respectively. A
large number of clones appear irrespective of the amounts of DNA
transfected and the culture conditions.
[0133] The clones coexpressing the E1 and E4 genes were screened by
a microinfection and microtitration technique. About one hundred
5606 clones are infected at a low moi (multiplicity of infection)
with a E1-E4-defective adenoviral vector (see, for example,
International Application WO 94/28152). A cytopathic effect is
observed 48 to 72 h after infection for 10 to 16% of them,
depending on the batch from which they originate. The cytopathic
effect in evidence of the multiplication of the doubly defective
viruses and reflects the capacity of the clone for.
trans-complementation. The titer of infectious viral particles is
estimated by a microwell titration technique on a permissive cell.
To this end, the viral particles are recovered from the infected
cultures by three cycles of freezing-thawing and the viral
supernatants to be titrated are diluted serially on the basis of
100 .mu.l per well and placed in contact with 4.times.10.sup.4 1653
cells. The viral titer is evaluated crudely by observation of the
cytopathic affect at the diff rent dilutions. The most productive
5606 clones (cytopathic effect at high dilutions) are selected for
a more accurate study of the yield in terms of pfu/ml and
ifu/ml.
[0134] The selected 5606 clones are cultured (5.times.10.sup.5
cells) and infected again with an E1-E4-adenoviral vector. It is
possible to use, for example, the vectors AdTG8595 and AdTG4651
from which the E1, E3 (nt 28592 to 30470) and E4 (nt 32994 to
34998) regions have been deleted, and comprising in place of E1 the
cassette for the expression of the gone of interest, MLP-LacZ [sic]
promoter and MLP-CFTR gone promoter, respectively. As before, the
viral supernatants are recovered and the titer in pfu/ml is
determined by infecting a permissive line (line 1653 or the 5606
productive clone) with suitable dilutions of viral superantant and
counting the lytic plaques in agar. Two clones, designated #5-19/1
and #5-38, give titers varying from 1 to 5.times.10.sup.9 pfu/ml
(titration performed on themselves). The yield of infectious
particles (ifu/ml) is evaluated by counting the infected cells
expressing the gone of interest. Thus, the viral superantants
obtained from the 5606 clones infected with AdTG8595 are titrated
with themselves and, 48 hours after infection, the adherent cells
are fixed (PBS buffer containing 2% of formaldehyde and 0.2% of
glutaraldehyde) and the production of .beta.-galactosidase
visualized with the dye X-Gal (1 mg/ml in PBS buffer to which 5 mM
K ferricyanide, 5 mM K ferrocyanide and 2 mM MgCl.sub.2 are added).
The titers obtained are between 10.sup.10 and 10.sup.11 ifu/ml.
[0135] Lastly, it is verified that the expression of the late viral
genes is not impaired, which might lead to a defect of assembly of
the viral particles. The production of fiber and penton in
determined by western blotting on the pellets of infected cells
recovered after the cycles of freezing-thawing. The analysis is
performed according to standard techniques on an aliquot of cell
extract corresponding to 3 .mu.g of proteins using antibodies
directed against the fiber (Henry et al., 1994, J. Virol. 68,
5239-5246) or the penton base (Boulanger et al., 1973, Bur. J.
Biochem. 39, 37-42), followed by peroxidase-labelled anti-rabbit
antibodies (Amersham, NA 934). Visualization takes place using the
ECL detection kit (Aersham, RPN 2106). The data show that the E1-
E4-adenoviruses generated from the two 5606 clones are capable of
producing the late proteins at a level approaching that obtained in
293 cells infected with an E1.sup.- virus.
[0136] In conclusion, the 5606 clones #5-19/1 and #5-38 constitute
E1 and E4 complementation lines capable of amplifying the doubly
defective vectors at a titer exceeding 1.times.10.sup.9 pfu/ml, a
titer compatible with a large-scale production.
EXAMPLE 4
[0137] Construction of the Complementation Line 9579 for the E1 and
E2A Functions, in which the Expression of E2A is Inducible with
tTA, the Latter Being Produced by the Line.
[0138] This example describes a complementation cell line
permitting the propagation of E1.sup.- and E2A.sup.- deficient
adenoviruses obtained by transfection of 293 cells with the vector
pTG9579 permitting a self-inducible expression of the DBP protein
according to the same principle as in the previous example.
[0139] 1. Construction of the Vector pTG9579
[0140] The vector pTG9579 in constructed in the following
manner:
[0141] The 3' end of the adenovirus 5 gone is isolated from a
preparation of genomic DNA digested with DraI-BsmBI. The
corresponding fragment is subcloned into the SmaI site of the
vector pUC19 (Gibco BRL) to give. the intermediate vector pTG9568.
The STOP codon of the DBP sequence in found to be destroyed by the
cleavage with DraI. It is restored by introduction between the
EcoRV and BamHI sites of pTG9568 of a fragment containing the
correct sequence obtained by PCR amplification from the genomic
template and the appropriate primers. pTG9571 is obtained.
[0142] Separately, the vector pUHD-10-3 is linearized with HindIII
and treated with the Klenow fragment, and the cassette for the
expression of the neo gene (Colbre-Garapin et al., 1981, J. Mol.
Biol. 150, 1-14) directed by the early promoter and the SV40 pA
signal are inserted. The vector thereby obtained, pTG9555, is
linearized with XbaI and then subjected to the action of the Klenow
fragment, before cloning the fragment carrying the DBP sequence
isolated from pTG9571 after cleavage with XhoI and EcoRI and
treatment with the Klenow fragment. Lastly, the HpaI-XhoI fragment
(Klenow) isolated from pTG4659 carrying the cassette for the
expression of the tTA gene is introduced into the XhoI site,
treated with the Klenow fragment, of the vector pTG9577 generated
in the previous step, to give pTG9579. To summarize, the latter
comprises:
[0143] a first cassette composed of the pCMV.sup.+ promoter
followed by the sequences coding for the tTA transactivator and the
SV40 virus polyA sequences,
[0144] a second cassette composed of the neo gene for resistance to
the antibiotic G418 under the control of the early promoter and the
SV40 virus pA sequences, and
[0145] a third cassette composed of the sequences coding for the
adenovirus 5 DBP protein under the control of the pCMV.sup.+
promoter and the SV40 virus pA sequences.
[0146] The production of a functional DBP protein by pTG9579 may be
verified by transient transfection into 293 cells. Briefly,
1.times.10.sup.6 cells are transfected with 5 .mu.g of vector by
the calcium phosphate method and then cultured in the presence (1
.mu.g/ml) or in the absence of tetracycline. 4 days after
transfection, the cells are recovered and the cell pellet is
treated with 100 .mu.l of lysis buffer (50 mM Tris-HCL [sic], 1 mM
MgCl.sub.2, 1 mM EDTA, 5 mM KCl, 150 mM NaCl and 1% Triton X-100)
in order to solubilize the proteins. 7 .mu.l of protein extract are
taken up in a Tris-glycine loading buffer containing 5% of
.beta.-mercaptoethanol and subjected to an 8-16% PAGE
electrophoresis under denaturing conditions. After transfer onto
nitrocellulose membranes, the DBP protein is visualized by western
blotting using an antibody specific for the adenoviral DBP protein
(Reich et al., 1983, Virology, 128, 480-484; for example the mouse
monoclonal antibody .alpha.72KB 6-8) and a peroxidase-labelled
sheep anti-mouse immunoglobulin antibody (Amersham; NA 931).
Visualization in performed with the ECL kit (Amershan, RPN 2106).
The results presented in FIG. 12 show that the DBP protein is
produced in 293 cells transiently transfected with the vector
pTG9579, and in a regulated manner since the expression in reduced
in the presence of tetracycline.
[0147] 2. Generation of the Complementation Cell Line 9579
[0148] 20 .mu.g of plasmid pTG9579 are used to transfect
3.times.30.sup.6 293 cells (Gibco BRL transfection kit; ref.
530-8120SA). The clones are selected in the presence of 600
.mu.g/ml of G418 and 1 .mu.g/al of tetracycline, and tested for
their capacity to produce the DBP protein by the western blotting
technique described above. 20% of then express detectable amounts
of the DBP, of which two, designated #7-44 and #7-32, at a level
close to that obtained in line gmDBP6 in the presence of
dexamethasone. The latter line constitutes the reference line for
complementation of the E2A function (Brough et al., 1992, Virol.
190, 624-634). It is derived from H*La cello transfected with an
expression cassette comprising the sequences coding for DBP,
directed by the dexamethasone-inducible MMTV promoter.
[0149] The DBP-positive clones are cultured and infected at an moi
.gtoreq.1 with the adenovirus H5d1802 which is defective for the
E2A function (Rice and Klessig, 1985, J. Virol. 56, 767-778). Two
days after infection, the viral supernatants are collected after
lysis of the cells by consecutive cycles of freezing-thawing,
diluted serially and titrated on the permissive line gmDBP6. The
presence of viral particles is observed in the supernatants
originating from the clones #7-44 and #7-32, showing their capacity
to complement the E2A function. The same type of experiment is
carried out with the vector pTG9542 (E1.sup.- (LacZ) E2A.sup.- and
E3.sup.-). Infectious particles are produced, showing that these
two clones are capable of propagating and amplifying doubly
defective vectors. Furthermore, these particles cannot be
propagated on line 293, confirming the E2A.sup.- phenotype and the
absence of revertants.
[0150] Amplification experiments conducted with the clone #7-44
infected with the AdTG9542 virus showed an amplification factor of
approximately 125 when the titration is performed 4 days after
infection.
* * * * *