U.S. patent application number 11/978617 was filed with the patent office on 2009-10-08 for circular dna molecule having a conditional origin of replication, process for their preparation and their use in gene therapy.
Invention is credited to Fabienne Soubrier.
Application Number | 20090252713 11/978617 |
Document ID | / |
Family ID | 32092417 |
Filed Date | 2009-10-08 |
United States Patent
Application |
20090252713 |
Kind Code |
A1 |
Soubrier; Fabienne |
October 8, 2009 |
CIRCULAR DNA MOLECULE HAVING A CONDITIONAL ORIGIN OF REPLICATION,
PROCESS FOR THEIR PREPARATION AND THEIR USE IN GENE THERAPY
Abstract
A circular DNA molecule, useful for gene therapy, comprising at
least one nucleic acid sequence of interest, characterised in that
the region allowing the replication thereof has an origin of
replication with a functionality in a host cell that requires the
presence of at least one specific protein foreign to said host
cell. A method for preparing same, cells incorporating said DNA
molecules and uses thereof in gene therapy are also described.
Inventors: |
Soubrier; Fabienne; (Thieis,
FR) |
Correspondence
Address: |
FINNEGAN, HENDERSON, FARABOW, GARRETT & DUNNER;LLP
901 NEW YORK AVENUE, NW
WASHINGTON
DC
20001-4413
US
|
Family ID: |
32092417 |
Appl. No.: |
11/978617 |
Filed: |
October 30, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11521412 |
Sep 15, 2006 |
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11978617 |
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10268948 |
Oct 11, 2002 |
7279313 |
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11521412 |
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09043193 |
Mar 13, 1998 |
6977174 |
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PCT/FR96/01414 |
Sep 13, 1996 |
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10268948 |
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Current U.S.
Class: |
424/93.21 ;
435/252.3; 435/455 |
Current CPC
Class: |
C12N 15/69 20130101;
C12N 15/74 20130101; A61P 7/00 20180101; A61K 48/00 20130101; A61P
7/04 20180101; A61P 9/00 20180101; A61P 9/10 20180101; A61P 21/00
20180101; C07K 14/501 20130101; A61P 31/14 20180101; A61P 25/28
20180101; A61P 31/12 20180101; A61P 35/00 20180101; A61P 25/00
20180101; A61P 25/16 20180101; C07K 14/82 20130101; A61P 31/18
20180101; C12N 15/70 20130101 |
Class at
Publication: |
424/93.21 ;
435/252.3; 435/455 |
International
Class: |
A61K 35/12 20060101
A61K035/12; C12N 1/21 20060101 C12N001/21; C12N 15/87 20060101
C12N015/87 |
Claims
1-18. (canceled)
19. A prokaryotic recombinant host cell comprising (a) a
heterologous replication initiation protein that activates a
conditional origin of replication, wherein the replication
initiation protein is encoded by gene pir 16; and (b) a gene
encoding a protein that is essential for host cell viability under
defined culture conditions, wherein the gene has a codon that is
recognized by a suppressor tRNA.
20. The prokaryotic recombinant host cell according to claim 19,
wherein the codon that is recognized by the suppressor tRNA is an
amber codon.
21. The prokaryotic recombinant host cell according to claim 19,
wherein the gene having the codon that is recognized by the
suppressor tRNA is a gene involved in the biosynthesis of a
metabolite or a catabolism gene.
22. The prokaryotic recombinant host cell according to claim 21,
wherein the metabolite is an amino acid.
23. The prokaryotic recombinant host cell according to claim 22,
wherein the amino acid is arginine.
24. The prokaryotic recombinant host cell according to claim 21,
wherein the gene having the codon that is recognized by the
suppressor tRNA is the argE gene.
25. The prokaryotic recombinant host cell according to claim 19,
further comprising an extrachromosomal DNA molecule, wherein the
extrachromosomal DNA molecule comprises a gene coding for a
suppressor tRNA.
26. The prokaryotic recombinant host cell according to claim 25,
wherein the suppressor tRNA is for an amber codon.
27. The prokaryotic recombinant host cell according to claim 19,
wherein the conditional origin of replication is from bacterial
plasmid R6K.
28. A method of expressing a gene of interest in an animal cell
comprising in vitro transfection of the animal cell with an
extrachromosomal DNA molecule comprising: (a) a gene of interest
operably linked to a promoter and a polyadenylation site; (b) a
conditional origin of replication whose functionality in a
prokaryotic host cell requires a replication initiating protein
that is specific for the origin of replication, foreign to the
prokaryotic host cell, and not encoded by the extrachromosomal DNA;
(c) a gene encoding a suppressor tRNA; and (d) a target for a
site-specific recombinase.
29. The method of claim 28, wherein the conditional origin of
replication is the .gamma. origin of replication of the plasmid
R6K.
30. The method of claim 28, wherein the gene encoding the
suppressor tRNA is for amber codons.
31. The method of claim 28, wherein the target for a site-specific
recombinase is a cer fragment from ColE1.
32. The method of claim 28, wherein the animal cell is a human
cell.
33. The method of claim 28, wherein the gene of interest encodes a
therapeutic protein.
34. The method of claim 33, wherein the method further comprises
administering the transfected animal cells to an animal.
35. A method of expressing a gene of interest in an animal cell
comprising injecting into an animal a composition comprising an
extrachromosomal DNA molecule comprising: (a) a gene of interest
operably linked to a promoter and a polyadenylation site; (b) a
conditional origin of replication whose functionality in a
prokaryotic host cell requires a replication initiating protein
that is specific for the origin of replication, foreign to the
prokaryotic host cell, and not encoded by the extrachromosomal DNA;
(c) a gene encoding a suppressor tRNA; and (d) a target for a
site-specific recombinase.
36. The method of claim 35, wherein the composition comprises a
cationic polymer or lipid.
37. The method of claim 35, wherein the conditional origin of
replication is the .gamma. origin of replication of the plasmid
R6K.
38. The method of claim 35, wherein the gene encoding the
suppressor tRNA is for amber codons.
39. The method of claim 35, wherein the target for a site-specific
recombinase is a cer fragment from ColE1.
40. The method of claim 35, wherein the animal cell is a human
cell.
41. The method of claim 35, wherein the gene of interest encodes a
therapeutic protein.
Description
[0001] This application is a continuation of application Ser. No.
11/521,412, filed Sep. 15, 2006, which is a continuation of
application Ser. No. 10/268,948, filed Oct. 11, 2002, which is a
continuation-in-part of application Ser. No. 09/043,193, filed Mar.
13, 1998 (now U.S. Pat. No. 6,977,174), which is a 371 of
application no. PCT/FR96/01414, filed Sep. 13, 1996, each of which
is incorporated by reference herein.
[0002] The present invention relates to a novel conditional
replication DNA molecule which can be used in gene therapy or for
the production of recombinant proteins.
[0003] Gene therapy consists in correcting a deficiency or an
anomaly by introducing genetic information into the affected organ
or cell. This information may be introduced either in vitro into a
cell extracted from the organ and then reinjected into the body, or
in vivo, directly into the target tissue. As a molecule of high
molecular weight and of negative charge, DNA has difficulty in
spontaneously crossing phospholipid cell membranes. Various vectors
are thus used in order to enable gene transfer to take place: viral
vectors, on the one hand, and natural or synthetic chemical and/or
biochemical vectors, on the other hand.
[0004] Viral vectors (retroviruses, adenoviruses, adeno-associated
viruses, etc.) are very effective, in particular for crossing
membranes, but present a certain number of risks such as
pathogenicity, recombination, replication, immunogenicity, etc.
[0005] Chemical and/or biochemical vectors allow these risks to be
avoided (for reviews, see Behr, 1993, Cotten and Wagner 1993).
These are, for example, cations (calcium phosphate, DEAE-dextran,
etc.) which act by forming precipitates with DNA, which may be
"phagocytosed" by the cells. They may also be liposomes in which
the DNA is incorporated and which fuse with the plasma membrane.
Synthetic gene-transfer vectors are generally lipids or cationic
polymers which complex the DNA and form with it a particle bearing
positive surface charges. As illustrations of vectors of this type,
mention may be made in particular of dioctadecylamidoglycylspermine
(DOGS, Transfectam.TM.) or
N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium (DOTMA,
Lipofectin.TM.).
[0006] However, the use of chemical and/or biochemical vectors or
naked DNA implies the possibility of producing large amounts of DNA
of pharmacological purity. The reason for this is that in gene
therapy techniques, the medicinal product consists of the DNA
itself and it is essential to be able to manufacture, in suitable
amounts, DNAs having properties which are appropriate for
therapeutic use in man.
[0007] In the case of non-viral vectorology, the vectors used are
plasmids of bacterial origin. The plasmids generally used in gene
therapy carry (i) an origin of replication, (ii) a marker gene such
as a gene for resistance to an antibiotic (kanamycin, ampicillin,
etc.) and (iii) one or more transgenes with sequences necessary for
their expression (enhancer(s), promoter(s), polyadenylation
sequences, etc.).
[0008] However, the technology currently available is not entirely
satisfactory.
[0009] On the one hand, the risk remains of dissemination in the
body. Thus, a bacterium which is present in the body can, at low
frequency, receive this plasmid. There is a greater likelihood of
this taking place if it involves an in vivo gene therapy treatment
in which the DNA may be disseminated in the body of the patient and
may come into contact with bacteria which infect this patient or
bacteria of the commensal flora. If the bacterium receiving the
plasmid is an enterobacterium, such as E. coli, this plasmid can be
replicated. Such an event then leads to dissemination of the
therapeutic gene. Insofar as the therapeutic genes used in gene
therapy treatments can code, for example, for a lymphokine, a
growth factor, an anti-oncogene or a protein whose function is
defective in the host and which thus makes it possible to correct a
genetic defect, the dissemination of some of these genes could have
unforeseeable and worrying effects (for example if a pathogenic
bacterium acquired a human growth factor gene).
[0010] On the other hand, the plasmids generally used in non-viral
gene therapy also possess a marker for resistance to an antibiotic
(ampicillin, kanamycin, etc.). The bacterium acquiring such a
plasmid thus has an undeniable selective advantage since any
antibiotic therapy, using an antibiotic from the same family as
that which selects the plasmid resistance gene, will lead to
selection of the plasmid in question. In this respect, ampicillin
belongs to the .alpha.-lactams, which is the family of antibiotics
which is most frequently used worldwide. The use in bacteria of
selection markers which are not antibiotic-resistance genes would
thus be particularly advantageous. This would avoid the selection
of bacteria which may have received a plasmid carrying such a
marker.
[0011] It is thus particularly important to seek to limit the
dissemination of therapeutic genes and resistance genes as much as
possible.
[0012] The subject of the present invention is specifically to
propose novel DNA molecules which can be used in gene therapy or
for the production of recombinant proteins in vitro and which
replicate only in cells which can complement certain functions of
these non-viral vectors.
[0013] The invention also relates to a particularly effective
method for preparing these DNA molecules.
[0014] The DNA molecules claimed have the advantage of removing the
risks associated with dissemination of the plasmid, such as (1)
replication and dissemination, which can lead to uncontrolled
overexpression of the therapeutic gene, (2) dissemination and
expression of resistance genes. The genetic information contained
in the DNA molecules according to the invention effectively
comprises the therapeutic gene(s) and the signals for regulating
its (their) expression, a functional conditional origin of
replication which greatly limits the host cell spectrum of this
plasmid, a selection marker of reduced size which is preferably
different from a gene which imparts resistance to an antibiotic
and, where appropriate, a DNA fragment which allows the resolution
of plasmid multimers. The probability of these molecules (and thus
the genetic information which they contain) being transferred to a
microorganism, and maintained stably, is very limited.
[0015] Lastly, the vectors according to the invention, also
referred to as miniplasmids on account of their circular structure,
their reduced size and their supercoiled form, have the following
additional advantages: on account of their size which is reduced in
comparison with the ColE1-derived plasmids used conventionally, the
DNA molecules according to the invention potentially have better in
vivo bioavailability. In particular, they have improved capacities
of cell penetration and distribution. Thus, it is acknowledged that
the diffusion coefficient in tissues is inversely proportional to
the molecular weight (Jain, 1987). Similarly, in the cell, high
molecular weight molecules have poorer permeability across the
plasma membrane. In addition, in order for the plasmid to pass into
the nucleus, which is essential for its expression, the high
molecular weight is also a disadvantage, the nuclear pores imposing
a size limit for diffusion into the nucleus (Landford et al.,
1986). The reduction in size of the non-therapeutic parts of the
DNA molecule (origin of replication and selection gene in
particular) according to the invention also makes it possible to
decrease the size of the DNA molecules. The part which allows the
replication and selection of this plasmid in the bacterium (1.1 kb)
is decreased by a factor of 3, counting, for example, 3 kb for the
origin of replication and the resistance marker vector part. This
decrease (i) in molecular weight and (ii) in negative charge
imparts improved tissue, cellular and nuclear bioavailability and
diffusion to the molecules of the invention.
[0016] More precisely, the present invention relates to a circular
DNA molecule, which is useful in gene therapy, this molecule
comprising at least one nucleic acid sequence of interest,
characterized in that the region which allows its replication
comprises an origin of replication whose functionality in a host
cell requires the presence of at least one specific protein which
is foreign to the said host cell.
[0017] This DNA molecule may be in single- or double-stranded form
and advantageously possesses a supercoiled form.
[0018] For the purposes of the present invention, the host cells
used can be of various origins. They can be eukaryotic or
prokaryotic cells. According to a preferred embodiment of the
invention, they are prokaryotic cells.
[0019] The replication of bacterial plasmids conventionally
requires the presence of at least one protein, which is coded for
by the host cell, of the RNA polymerase, Rnase, DNA polymerase,
etc. type. For the reasons already explained above, it is not
possible to overcome entirely, with this type of replication, any
possible risks of dissemination in the treated organism.
Advantageously, the functionality of the origin of replication of
the DNA molecule according to the invention requires the presence
of a specific protein which is foreign to the host cell. The
significance of this characteristic is to reduce the host spectrum
of the claimed plasmid to specific strains that express this
initiator protein. The DNA molecule developed within the context of
the present invention thus advantageously possesses a so-called
conditional origin of replication.
[0020] The conditional origin of replication used according to the
present invention may originate from plasmids or bacteriophages
which share the following characteristics: they contain in their
origin of replication repeat sequences, or iterons, and they code
for at least one replication-initiating protein (Rep) which is
specific to them. By way of example, mention may be made of the
conditional replication systems of the following plasmids and
bacteriophages:
TABLE-US-00001 specific initiator plasmid or bacteriophage protein
RK2 (Stalker et al., 1981) TrfA R1 (Ryder et al., 1981) RepA pSC101
(Vocke and Bastia, 1983) RepA F (Murotsu et al., 1981) protein E
Rts1 (Itoh et al., 1982, 1987) RepA RSF1010 (Miao et al., 1995)
RepC P1 (Abeles et al., 1984) RepA P4 (Flensburg and Calendar,
1987) alpha protein lambda (Moore et al., 1981) protein O phi 82
(Moore et al., 1981) protein O from phi 82 phi 80 protein O from
phi 80
[0021] According to a preferred embodiment of the invention, the
origin of replication used in the DNA molecules claimed is derived
from a natural E. Coli plasmid referred to as R6K.
[0022] The replication functions of R6K are grouped together in a
5.5 kbp DNA fragment (FIG. 1) comprising 3 origins of replication
.alpha., .beta., and .gamma. (.gamma. and .alpha. providing 90% of
the replication) and an operon coding for the .pi.
replication-initiator proteins and the protein Bis. The minimum
amount of genetic information required to maintain this plasmid at
its characteristic number of copies (15 copies per genome) is
contained in two elements: the 400 bp of ori .gamma. and the gene
pir, whose product is the .pi. initiator protein.
[0023] Ori .gamma. may be divided into two functional parts: the
core part and the activator element (FIG. 1). The core part, which
is essential for replication, contains the iterons (7 direct
repeats of 22 bp) to which the .pi. protein represented in SEQ ID
No. 1 becomes bound, and flanking segments, which are targets of
the host proteins (IHF, DnaA).
[0024] According to a preferred mode of the invention, the origin
of replication of the vector claimed consists entirely or partially
of this .gamma. origin of replication of the plasmid R6K and more
preferably, entirely or partially of SEQ ID No. 1 or one of its
derivatives.
[0025] For the purposes of the present invention, the term
derivative denotes any sequence which differs from the sequence
considered on account of degeneracy of the genetic code, obtained
by one or more modifications of genetic and/or chemical nature, as
well as any sequence which hybridizes with these sequences or
fragments thereof and whose product possesses the activity
indicated with regard to the replication-initiator protein .pi..
The term modification of the genetic and/or chemical nature may be
understood to refer to any mutation, substitution, deletion,
addition and/or modification of one or more residues. The term
derivative also comprises the sequences homologous with the
sequence considered, derived from other cellular sources and in
particular cells of human origin, or from other organisms, and
possessing an activity of the same type. Such homologous sequences
may be obtained by hybridization experiments. The hybridizations
may be performed starting with nucleic acid libraries, using the
native sequence or a fragment thereof as probe, under conventional
conditions of stringency (Maniatis et al., cf. General techniques
of molecular biology), or, preferably, under conditions of high
stringency.
[0026] The origin of replication described above, which has the
advantage of being of very limited size, is functional solely in
the presence of a specific initiator protein, protein Pi, produced
by the gene pir (SEQ ID No. 2). Since this protein can act in
trans, it is possible to physically dissociate the ori gamma from
the pir gene, which may be introduced into the genome of the cell
which is chosen as the specific host for these plasmids. Mutations
in .pi. may alter its inhibitory functions (Inuzuka and Wada, 1985)
and lead to an increase in the number of copies of the R6K
derivatives, up to more than 10 times the initial number of copies.
These substitutions are all within a domain of 40 amino acids,
which therefore appears to be responsible for the control by .pi.
of the number of plasmid copies (FIG. 2).
[0027] According to an advantageous embodiment of the present
invention, the .pi. protein, expressed in the host cell, results
from the expression of the gene represented in SEQ ID No. 2 or one
of its derivatives as defined above and more particularly of the
gene pir 116 which comprises a mutation when compared with the pir
gene. This mutation corresponds to the replacement of a proline by
a leucine. In this context, the number of copies of the R6K
derivatives is about 250 copies per genome.
[0028] Besides a conditional origin of replication as defined
above, the DNA molecules claimed contain a region comprising one
(or more) gene(s) which make it possible to ensure selection of the
DNA molecule in the chosen host.
[0029] This may be a conventional marker of gene type which imparts
resistance to an antibiotic, such as kanamycin, ampicillin,
chloramphenicol, streptomycin, spectinomycin, lividomycin or the
like.
[0030] However, according to a preferred embodiment of the
invention, this region is different from a gene which imparts
resistance to an antibiotic. It may thus be a gene whose product is
essential for the viability of the host envisaged, under defined
culturing conditions. It may be, for example: [0031] a gene coding
for a suppressor tRNA, of natural or synthetic origin. This is,
more preferably, an amber codon tRNA (TAG) [0032] a gene whose
product is necessary for metabolism of the cell, under certain
culturing conditions, namely a gene involved in the biosynthesis of
a metabolite (amino acid, vitamin, etc.), or a catabolism gene
which makes it possible to assimilate a substance present in the
culture medium (specific nitrogen or carbon source), etc.
[0033] According to a preferred mode of the invention, this region
contains an expression cassette of a gene coding for a suppressor
tRNA for specific codons. This latter may be chosen, in particular,
from those coding for phenylalanine, cysteine, proline, alanine and
histidine bases. It is more particularly a suppressor tRNA for
amber codons (TAG).
[0034] In this particular case, the system used to select, in the
host cells, the DNA molecules which are the subject of the present
invention includes two elements: 1) on the DNA molecule, a gene
coding for a suppressor transfer RNA for the amber codon (TAG)
which constitutes the selection marker, known as (sup) gene and 2)
a specific host, one of whose genes, which is essential under
certain culture conditions, contains an amber TAG codon. This cell
may grow, under the culture conditions for which the product of the
gene containing the TAG codon is essential, only if the plasmid
allowing the expression of sup is present in the cell. The culture
conditions thus constitute the pressure for selection of the DNA
molecule. The sup genes used may be of natural origin (Glass et
al., 1982) or may originate from a synthetic construction (Normanly
et al., 1986, Kleina et al., 1990).
[0035] Such a system offers great flexibility insofar as, depending
on the gene containing an amber mutation, it is possible to
determine various selective media. In the bacterium Lactococcus
lactis for example, the amber codon is located in a purine
biosynthesis gene. This allows the selection of the plasmid
carrying the gene coding for the suppressor tRNA when the bacteria
multiply in milk. Such a marker has the advantage of being very
small and of containing no "foreign" sequences, originating from
phages or transposons.
[0036] According to a particular embodiment of the invention, the
DNA molecule also comprises a DNA fragment, the target for
site-specific recombinases, which allows the resolution of plasmid
multimers.
[0037] Thus, such a fragment, introduced on to a DNA molecule which
is circular and whose origin of replication is, for example, ori
gamma, allows the resolution of multimers of such a plasmid. Such
multimers are observed, in particular, when the DNA molecule is
prepared in a strain carrying a mutated allele of pir, such as
pir-116, which makes it possible to increase the number of copies
of the R6K derivatives.
[0038] This recombination may be achieved by means of various
systems which entail site-specific recombination between sequences.
More preferably, the site-specific recombination of the invention
is obtained by means of specific intramolecular recombination
sequences which are capable of recombining with each other in the
presence of specific proteins, generally referred to as
recombinases. In this specific case, these are the recombinases
XerC and XerD. For this reason, the DNA molecules according to the
invention generally also comprise a sequence which allows this
site-specific recombination. The specific recombination system
present in the genetic constructions according to the invention
(recombinases and specific recognition site) may be of different
origins. In particular, the specific sequences and the recombinases
used may belong to different structural classes, and in particular
to the transposon Tn3 resolvase family or to the bacteriophage
lambda integrase family. Among the recombinases belonging to the
transposon Tn3 family, mention may be made in particular of the
resolvase of transposon Tn3 or of transposons Tn21 and Tn522 (Stark
et al., 1992); the Gin invertase of bacteriophage mu or
alternatively plasmid resolvases, such as that of the par fragment
of RP4 (Abert et al., Mol. Microbiol. 12 (1994) 131). Among the
recombinases belonging to the bacteriophage e integrase family,
mention may be made in particular of the integrase of the phages
lambda (Landy et al., Science 197 (1977) 1147), P22 and .sub.--80
(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 phage P1, the integrase of plasmid pSAM2 (EP 350 341)
or alternatively the FLP recombinase of the 21 plasmid and the XerC
and XerD recombinases from E. coli.
[0039] Preferably, the DNA molecules which form the subject of the
present invention contain the fragment cer from the natural E. coli
plasmid ColE1. The cer fragment used is a 382 bp HpaII fragment
from ColE1 which has been shown to bring about, in cis, the
resolution of plasmid multimers (Summers et al., 1984; Leung et
al., 1985). It is also possible to use a HpaII-TaqI fragment of
smaller size (280 bp) or a smaller fragment (about 220 bp),
contained in the HpaII fragment, which fragments possess the same
properties (Summers and Sherratt, 1988). This resolution takes
place by way of a specific intramolecular recombination, which
involves four proteins encoded by the genome of E. coli: ArgR,
PepA, XerC and XerD (Stirling et al., 1988, 1989; Colloms et al.,
1990, Blakely et al., 1993).
[0040] In this respect, it is particularly advantageous to use all
or part of the cer fragment of ColE1 or one of its derivatives as
defined above.
[0041] According to an implementation variant, the DNA molecules of
the invention may also comprise a sequence capable of interacting
specifically with a ligand. Preferably, this is a sequence capable
of forming, by hybridization, a triple helix with a specific
oligonucleotide. This sequence thus makes it possible to purify the
molecules of the invention by selective hybridization with a
complementary oligonucleotide immobilized on a support (see
application WO 96/18744). The sequence can be positioned at any
site in the DNA molecule of the invention, provided that it does
not affect the functionality of the gene of interest and of the
origin of replication.
[0042] As a DNA molecule representative of the present invention,
the plasmid pXL2774 and its derivatives may be claimed most
particularly. For the purposes of the invention, the term
derivative is understood to refer to any construction derived from
pXL2774 and containing one or more genes of interest other than the
luciferase gene. Mention may also be made of the plasmids pXL3029
and 3030 containing an expression cassette of a therapeutic gene
and a sequence capable of interacting specifically with a
ligand.
[0043] The present invention also relates to the development of a
process for the construction of specific host cells, which are
particularly effective for the production of these therapeutic DNA
molecules.
[0044] Another subject of the present invention relates to a
process for the production of a circular DNA molecule,
characterized in that a host cell is cultured containing at least
one DNA molecule as defined above and a protein, which may or may
not be expressed in situ, which conditions the functionality of the
origin of replication of the said DNA molecule, which is specific
and which is foreign to the said host cell, under conditions which
allow the selection of host cells transformed by the said DNA
molecules.
[0045] More preferably, the protein which conditions the
functionality of the origin of replication of the DNA molecule is
expressed in situ from a corresponding gene. The gene coding for
the replication-initiating protein may be carried by a subsidiary
replicon, which is compatible with the derivatives of the
conditional origin of replication used or which may be introduced
into the genome of the host cell by recombination, by means of a
transposon, a bacteriophage or any other vector. In the particular
case in which the gene expressing the protein is placed on a
subsidiary replicon, the latter also contains a promoter region for
functional transcription in the cell, as well as a region which is
located at the 3' end and which specifies a transcription
termination signal. As regards the promoter region, this may be a
promoter region which is naturally responsible for expressing the
gene under consideration when the latter is capable of functioning
in the cell. It may also be a case of regions of different origin
(responsible for expressing other proteins), or even of synthetic
origin. In particular, it may be a case of promoter sequences for
prokaryotic or bacteriophage genes. For example, it may be a case
of promoter sequences obtained from the cell genome.
[0046] As genes coding for the replication-initiating protein, use
may be made either of wild-type genes or of mutated alleles which
make it possible to obtain an increased number of copies of the
plasmids (or derivatives) specific for the initiator protein which
conditions the functionality of the origin of replication used in
the DNA molecule.
[0047] Such mutants have been described in particular for the
plasmids R6K (Inuzuka and Wada, 1985; Greener et al., (1990), Rts1
(Terawaki and Itoh, 1985, Terawaki et al., 1990; Zeng et al.,
1990), F (Seelke et al., 1982; Helsberg et al., 1985; Kawasaki et
al., 1991), RK2 (Durland et al., 1990; Haugan et al., 1992, 1995),
pSC101 (Xia et al., 1991; Goebel et al., 1991; Fang et al.,
1993).
[0048] In the particular case in which the DNA molecule used
possesses an origin of replication derived from the plasmid R6K,
the initiator protein is a derivative of the .pi. protein of this
same plasmid. It is particularly advantageous to express a mutated
form of this protein which is capable of increasing the number of
initial copies appreciably. To do this, the gene incorporated into
the host cell is preferably represented by all or part of the
sequence represented in SEQ ID No. 2 or one of its derivatives and
more preferably by the pir 116 gene. The associated mutation
corresponds to the replacement of a proline by a leucine. According
to a particular embodiment of the invention, this pir 116 gene is
directly incorporated into the host cell genome.
[0049] Advantageously, one of the genes of the specific host cell,
which is essential under the culture conditions chosen, contains a
specific codon which is recognizable by the selected suppressor
tRNA in the DNA molecule. According to a preferred mode of the
invention, this is an amber TAG codon. In this particular case, the
cell may grow, under culture conditions for which the product of
the gene containing the TAG codon is essential, only if the plasmid
allowing the expression of sup is present in the host cell. The
culture conditions thus constitute the pressure for selection of
the DNA molecule.
[0050] Preferably, the gene containing the amber codon is a gene
involved in the biosynthesis of an amino acid, arginine. This gene,
argE, codes for an N-acetylomithinase (Meinnel et al., 1992) and in
this case contains a TAG codon corresponding to a point mutation
Gln-53 (CAG)->TAG; the pressure for selection of the plasmid
carrying the sup gene is then provided by culturing in minimal M9
medium (Maniatis et al., 1989). However, this could also be, for
example, a gene for biosynthesis of a vitamin or a nucleic acid
base, or alternatively a gene which allows a specific nitrogen or
carbon source to be used or any other gene whose functionality is
essential for cellular viability under the chosen culture
conditions.
[0051] The host cell is preferably chosen from E. coli strains and
is more preferably represented by the strain E. coli XAC-1.
[0052] According to a specific embodiment of the invention, the
host cell used in the claimed process is a cell of the E. coli
strain XAC-1, containing the pir 116 gene in its genome and
transformed by the plasmid pXL2774 or one of its derivatives.
[0053] According to an advantageous variant of the invention, the
host cell used in the process claimed is a prokaryotic cell in
which the endA1 gene or a homologous gene is inactivated. The endA
gene codes for endonuclease I of E. coli. This periplasmic enzyme
has a non-specific activity of cleaving double-stranded DNA
(Lehman, I. R., G. G. Roussos and E. A. Pratt (1962) J. Biol. Chem.
237: 819-828; Wright M. (1971) J. Bacteriol. 107: 87-94). A study
carried out on various strains of Escherichia coli (wild-type or
endA) showed that the degradation of plasmid DNA incubated in
extracts of these bacterial strains existed in the endA+ strains
but not in the endA mutants. (Wnendt S. (1994) BioTechniques 17:
270-272). The quality of the plasmid DNA isolated from endA+
strains or from endA mutants was studied by the company Promega
using their purification system (Shoenfeld, T., J. Mendez, D.
Storts, E. Portman, B..dagger.Patterson, J. Frederiksen and C.
Smith. 1995. Effects of bacterial strains carrying the endA1
genotype on DNA quality isolated with Wizard plasmid purification
systems. Promega notes 53). Their conclusion is as follows: the
quality of the DNA prepared from endA mutants is, overall, better
than that of DNA prepared in the endA+ strains tested.
[0054] The quality of the plasmid DNA preparations is thus affected
by any contamination with this endonuclease (relatively long-term
degradation of the DNA).
[0055] The deletion or mutation of the endA gene can be envisaged
without difficulty insofar as the mutants no longer having this
endonuclease activity behave on the whole like wild-type bacteria
(Durwald, H. and H. Hoffmann-Berling (1968) J. Mol. Biol. 34:
331-346).
[0056] The endA1 gene can be inactivated by mutation, total or
partial deletion, disruption, etc. Inactivation of the endA gene of
the E. coli strain chosen to produce the pCOR plasmids can be
achieved more particularly by transferring, by means of the P1
bacteriophage, the .DELTA.endA::Tc.sup.R deletion described by
Cherepanov and Wackernagel (Cherepanov, P. P. and W. Wackemagel.
1995. Gene disruption in Escherichia coli: Tc.sup.R and Km.sup.R
cassettes with the option of Flp-catalyzed excision of the
antibiotic-resistance determinant. Gene 158:9-14) or by exchanging
the wild-type allele present in the genome of the bacterium of
interest with a mutated or deleted allele of endA, by homologous
recombination. The use of this type of strain in the context of the
present invention makes it possible advantageously to improve the
quality of the DNA produced.
[0057] The invention also relates to any recombinant cell
containing a DNA molecule as defined above. This may be a cell of
various origins, of eukaryotic, prokaryotic, etc. type.
[0058] According to another embodiment of the invention, the E.
coli XAC-1 host cell used in the process claimed is designated
TEX1, and comprises a traD gene, or a homologous gene thereof,
inactivated to abolish F' transfer. The traD is at the 5' end of
one of the tra operons and encodes a 81.7 kDa membrane protein that
is directly involved in DNA transfer and DNA metabolism (Frost et
al., Microbiology Reviews, 1994, 58: 162-210). traD mutants do not
transfer DNA (Panicker et al., J. Bacteriol., 1985, 162:584-590).
The episomal traD gene may be inactivated by mutation, total or
partial deletion, or disruption using methods well known to those
of skill in the art (See Example 9). One method of inactivating
this gene is described in Example 1, and the resulting E. coli
XAC-1 pir116 endA.sup.- traD.sup.- strain so obtained is designated
TEX1 (Soubrier et al., Gene Therapy, 1999, 6: 1482-1488).
[0059] According to one embodiment of the invention, the host cell
used in the claimed process is a cell of the E. coli strain XAC-1,
containing the pir116 mutation combined with the pir42 mutation.
The pir116 and pir42 mutations affect different domains of the n
protein. The pir116 mutation affects the copy number control
region, whereas the pir42 mutation affects the putative leucine
zipper motif, as displayed in FIG. 11. The nucleotide and amino
acid sequences of the pir gene containing the pir116 and pir42
mutations are set forth in FIG. 12 and SEQ ID NOs: 21 and 22,
respectively. The pir42 mutation comprises a C to T transition at
position 124 from the N-terminal methionine (as referenced by the
first amino acid of the sequence), and thus results in substitution
of the proline at position 42 by a leucine. The pir42 mutation was
described by Miron et al. (Proc Natl Acad Sci USA, 1994. 91(14): p.
6438-42; EMBO J, 1992. 11(3): p. 1205-16), and was reported to
increase the copy number of an "ori gamma R6K-Km.sup.R-pir42"
plasmid by 2.5 fold as compared to the same plasmid harboring the
wild-type pir gene. However the pir42 mutation was never used or
described in combination with the pir116 mutation and while other
mutations such as cop21 in the pir gene combined with the pir116 do
not exhibit an increase of the plasmid copy number, combination of
the pir116 and pir42 mutations in a E. coli XAC-1 endA.sup.-
traD.sup.- strain surprisingly showed a significant increase of the
plasmid copy number. Applicants have thus shown unexpected results
of this combination in terms of copy number of the plasmids
produced in E. coli host strains comprising the mutated pir116 and
pir42 gene as compared with strains harboring pir116 alone, or in a
host cell comprising the pir116 mutation combined with another
mutation of the pir gene, such as the mutation cop21 (Inuzuka et
al., FEBS Lett, 1988. 228(1): p. 7-11). For example, E. coli
TEX1pir42 (-XAC-1 endA.sup.- traD.sup.- pir116 pir42) exhibited a
2-5 fold increase in the number of plasmid, as compared to pir116
strain, or strains comprising combined pir116 and cop21 mutations
(See Example 11).
[0060] According to another embodiment of the present invention,
the host cell used in the process claimed is a prokaryotic host
cell in which the recA gene or a homologous gene has been
inactivated. Preferably, the host cell according to the present
invention is E. coli strain XAC-1 comprising mutations pir116,
pir42, endA.sup.-, traD.sup.-, recA.sup.- Such a strain is
designated TEX2pir42. recA may be inactivated by methods well known
to those in the art. recA encodes a major recombination protein and
mutations in this gene reduce the frequency of
recombination-mediated alteration in plasmids and intramolecular
recombination that could lead to the multimerization of plasmids.
As described in Example 12, a deleted recA gene containing 3
translation stop codons (one in each frame) at its 5' end may be
obtained by PCR. The resulting inactivated gene was then introduced
by gene replacement into TEX1 genome (Example 12.1).
[0061] These cells are obtained by any technique known to those
skilled in the art which allows the introduction of the said
plasmid into a given cell. Such a technique may be, in particular,
transformation, electroporation, conjugation, fusion of protoplasts
or any other technique known to those skilled in the art.
[0062] Strain TEX1 was deposited under the terms of the Budapest
Treaty with the Collection Nationale De Cultures De
Micro-organismes (CNCM), Institut Pasteur, 28, rue Dr. Roux, 75724
Paris Cedex 15, France, on Dec. 10, 2004, under accession no. CNCM
I-3343.
[0063] Strain TEX2pir42 was deposited under the terms of the
Budapest Treaty with the Collection Nationale De Cultures De
Micro-organismes (CNCM), Institut Pasteur, 28, rue Dr. Roux, 75724
Paris Cedex 15, France, on Oct. 10, 2003, under accession no. CNCM
I-3109.
[0064] Strain TEX2 was deposited under the terms of the Budapest
Treaty with the Collection National De Cultures De Micro-organismes
(CNCM), Institut Pasteur, 28, rue Dr. Roux, 75724 Paris Cedex 15,
France, on Dec. 10, 2004, under accession no. CNCM 1-3344.
[0065] Strain XAC1pir116 was deposited under the terms of the
Budapest Treaty with the Collection Nationale De Cultures De
Micro-organismes (CNCM), Institut Pasteur, 28, rue Dr. Roux, 75724
Paris Cedex 15, France, on Oct. 10, 2003, under accession no. CNCM
I-3108.
[0066] The DNA molecules according to the invention may be used in
any application of vaccination or of gene and cell therapy for
transferring a gene to a given cell, tissue or organism, or for the
production of recombinant proteins in vitro.
[0067] In particular, they may be used for direct in vivo
administration or for the modification of cells in vitro or ex
vivo, for the purpose of implanting them into a patient.
[0068] In this respect, another subject of the present invention
relates to any pharmaceutical composition comprising at least one
DNA molecule as defined above. This molecule may or may not be
associated therein with a chemical and/or biochemical transfection
vector. This may in particular involve cations (calcium phosphate,
DEAE-dextran, etc.) or liposomes. The associated synthetic vectors
may be cationic polymers or lipids. Examples of such vectors which
may be mentioned are DOGS (Transfectam.TM.) or DOTMA
(Lipofectin.TM.).
[0069] The pharmaceutical compositions according to the invention
may be formulated for the purpose of topical, oral, parenteral,
intranasal, intravenous, intramuscular, subcutaneous, intraocular,
or transdermal administrations. The claimed plasmid is preferably
used in an injectable form or in application. It may be mixed with
any vehicle which is pharmaceutically acceptable for an injectable
formulation, in particular for a direct injection to the site to be
treated. This may involve, in particular, sterile, isotonic
solutions or dry compositions, in particular freeze-dried
compositions, which, by addition, depending on the case, of
sterilized water or of physiological saline, allow injectable
solutions to be made up. This may in particular involve Tris or PBS
buffers diluted in glucose or in sodium chloride. A direct
injection into the affected region of the patient is advantageous
since it allows the therapeutic effect to be concentrated at the
level of the affected tissues. The doses used may be adapted as a
function of various parameters, and in particular as a function of
the gene, the vector, the mode of administration used, the
pathology concerned or the desired duration of the treatment.
[0070] The DNA molecules of the invention may contain one or more
genes of interest, that is to say one or more nucleic acids
(synthetic or semi-synthetic DNA, gDNA, cDNA, etc.) whose
transcription and, possibly, whose translation in the target cell
generate products of therapeutic, vaccinal, agronomic or veterinary
interest.
[0071] Among the genes of therapeutic interest which may be
mentioned more particularly are genes coding for enzymes, blood
derivatives, hormones and lymphokines: interleukins, interferons,
TNF, etc. (FR 92/03120), growth factors, neurotransmitters or their
precursors or synthetic enzymes, and trophic factors (BDNF, CNTF,
NGF, IGF, GMF, aFGF, bFGF, NT3, NT5, VEGF-B, VEGF-C etc.;
apolipoproteins: ApoAI, ApoAIV, ApoE, etc. (FR 93/05125),
dystrophin or a minidystrophin (FR 91/11947), tumour-suppressing
genes: p53, Rb, Rap1A, DCC, k-rev, etc. (FR 93/04745), genes coding
for factors involved in coagulation: factors VII, VIII, IX, etc.,
suicide genes: thymidine kinase, cytosine deaminase, etc.; or
alternatively all or part of a natural or artificial immunoglobulin
(Fab, ScFv, etc.), an RNA ligand (WO 91/19813), etc. The
therapeutic gene may also be an antisense sequence or gene, 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 which are complementary to cellular mRNAs and thus block
their translation into protein, according to the technique
described in patent EP 140,308.
[0072] The gene of interest may also be a vaccinating gene, that is
to say a gene coding for an antigenic peptide, capable of
generating an immune response in man or animals, for the purpose of
producing vaccines. These antigenic peptides may in particular be
specific antigenic peptides of Epstein-Barr virus, HIV virus,
hepatitis B virus (EP 185,573), or pseudorabies virus, or
alternatively specific antigenic peptides of tumours. (EP
259,212).
[0073] Generally, in the DNA molecules of the invention, the gene
of therapeutic, vaccinal, agronomic or veterinary interest also
contains a promoter region for functional transcription in the
target organism or cell, as well as a region located at the 3' end
which specifies a transcription termination signal and a
polyadenylation site. As regards the promoter region, it may be a
promoter region naturally responsible for expression of the gene
under consideration when this region is capable of functioning in
the cell or the organism concerned. The promoter regions may also
be regions of different origin (responsible for the expression of
other proteins) or even of synthetic origin. In particular, they
may be promoter sequences from eukaryotic or viral genes. For
example, they may be promoter sequences obtained from the genome of
the target cell. Among the eukaryotic promoters which may be used
are any promoter or derived sequence which stimulates or suppresses
the transcription of a gene in a specific or non-specific,
inducible or non-inducible, strong or weak manner. The eukaryotic
promoters may in particular be ubiquitous promoters (promoters of
the genes for HPRT, PGK, .alpha.-actin, tubulin, etc.),
intermediate filament promoters (promoters of the genes for GFAP,
desmin, vimentin, neurofilaments, keratin, etc.), therapeutic gene
promoters (for example the promoters of the genes for MDR, CFTR,
factor VIII, ApoAI, etc.) tissue-specific promoters (promoters of
the genes for pyruvate kinase, villin, intestinal fatty
acid-binding protein, {acute over (.alpha.)}-actin of smooth
muscle, etc.) or alternatively promoters which respond to a
stimulus (steroid hormone receptor, retinoic acid receptor, etc.).
Similarly, they may be promoter sequences obtained from the genome
of a virus, such as, for example, the promoters of the adenovirus
E1A and MLP genes, the CMV early promoter or alternatively the LTR
promoter of RSV, etc. In addition, these promoter regions may be
modified by addition of activating or regulatory sequences or
sequences which allow tissue-specific expression or expression
which is predominantly tissue-specific.
[0074] Moreover, the gene of interest may also contain a signal
sequence which directs the synthesized product into the secretory
pathways of the target cell. This signal sequence may be the
natural signal sequence of the synthesized product, but it may also
be any other functional signal sequence or an artificial signal
sequence.
[0075] Depending on the gene of interest, the DNA molecules of the
invention may be used for the treatment or prevention of several
pathologies, including genetic diseases (dystrophy, cystic
fibrosis, etc.), neurodegenerative diseases (Alzheimer's disease,
Parkinson's disease, ALS, etc.), cancers, pathologies associated
with coagulation disorders or with dyslipoproteinaemias,
pathologies associated with viral infections (hepatitis, AIDS,
etc.), or in the agronomic and veterinary fields, etc.
[0076] Moreover, the present invention also relates to the use of
conditional replication DNA molecules for the production of
recombinant proteins. Bacteria can be used to produce proteins of
various origins, eukaryotic or prokaryotic. Among the bacteria, E.
coli constitutes the organism of choice for expressing heterologous
genes on account of its ease of manipulation, the large number of
expression systems available and the large amounts of proteins
which can be obtained. It is understood that the system of the
invention can be used in other organisms, the tropism being
determined by the nature of the origin of replication, as indicated
above. For this use, the nucleic acid sequence of interest
comprises a coding region under the control of expression signals
that are appropriate for the host chosen, in particular a
prokaryotic host. These may be, for example, Plac, Ptrp, PT7, Ptrc,
Ptac, PL or PR promoters, the Shine-Dalgarno sequence, etc. (this
set constitutes the expression cassette). The nucleic acid sequence
of interest can be any sequence coding for a protein which is of
value in the fields of pharmacy, agri-foods, chemistry or
agrochemistry. This may be a structural gene, a complementary DNA
sequence, a synthetic or semi-synthetic sequence, etc.
[0077] The expression cassette can be introduced onto the
conditional replication vector which is the subject of the
invention, thus constituting a conditional replication vector which
allows the expression of proteins of interest in E. coli. This
vector has several advantages: no use of antibiotic to select it in
the bacterium (reduced cost, no need for a study regarding the
presence of antibiotic or of potentially toxic derived products in
the finished product), virtually no probability of dissemination of
the plasmid in nature (origin of conditional replication), possible
fermentation in entirely defined medium. The examples given show
the advantageous properties of these conditional vectors for the
production of recombinant proteins.
[0078] The present invention will be described more fully with the
aid of the examples which follow, which should be considered as
non-limiting illustrations.
BRIEF DESCRIPTION OF THE DRAWINGS
[0079] FIG. 1: Functional organization of the region of R6K
involved in replication.
[0080] FIG. 2: Organization of the functional domains of the .pi.
protein of the plasmid R6k.
[0081] FIG. 3: Representation of the protocol for introducting the
pir gene into the genome of E. coli XAC1.
[0082] FIG. 4: Construction scheme for vectors pXL2666, 2730 and
2754.
[0083] FIG. 5: Construction of pXL2774.
[0084] FIG. 6: Growth and production kinetics in a 2 L
fermenter.
[0085] FIG. 7: Growth and production kinetics in an 800 L
fermenter.
[0086] FIG. 8: Construction of pXL3056.
[0087] FIG. 9: Visualization of the aFGF protein produced by E.
coli XAC-1pir-116 (pXL3056+PT7pol23) after induction. The denatured
total cell extracts are deposited on 12.5%-SDS polyacrylamide gel.
M: molecular mass marker (Biorad, Low range). Each band is
identified by an arrow and a figure which indicates its mass in
kDaltons. 1: XAC-1pir-116 (pXL3056+pUC4K) not induced; 2:
XAC-1pir-116 (pXL3056+pUC4K) induced at 42.degree. C.; 3:
XAC-1pir-116 (pXL3056+PT7pol23) clone 1, not induced; 4:
XAC-1pir-116 (pXL3056+PT7pol23) clone 1, induced at 42.degree. C.;
5: XAC-1pir-116 (pXL3056+PT7pol23) clone 2, not induced; 6:
XAC-1pir-116 (pXL3056+PT7pol23) clone 2, induced at 42.degree. C.;
t1: 1 .mu.g of purified aFGF; t4: 4 .mu.g of purified aFGF.
[0088] FIG. 10: Construction scheme for vectors pXL3029 and
pXL3030.
[0089] FIG. 11: Schematic representation of the functional domains
of R6K .pi. initiator proteins.
[0090] FIG. 12: Nucleotide and amino acid sequences of the pir gene
comprising the pir116 and pir42 mutations.
[0091] FIG. 13: Construction of pir116-pir42 suicide vector for
homologous recombination.
[0092] FIG. 14: Schematic representation of the PCR products
obtained when amplifying the region uidA::pir116+/-pir42.
[0093] FIG. 15: Agarose gel electrophoresis showing the topology of
pCOR plasmid pXL3179 produced in TEX1 or TEX1pir42.
[0094] FIG. 16: Schematic representation of the pXL3749 suicide
plasmid carrying pir116-co 21 gene.
[0095] FIG. 17: Agarose gel electrophoresis showing the plasmid
copy number of pXL2979 when produced in E. coli host cell TEX1cop21
(lines 1-4), in E. coli host cell XAC1pir (lines 5-8), in E. coli
TEX1 (lines 9-12).
[0096] FIG. 18: Representation of the cloning strategy for the
construction of the recA-suicide vector.
[0097] FIG. 19: Schematic representation of the PCR products
obtained when amplifying regions of E. coli TEX2 strain.
[0098] FIG. 20: Agarose gel electrophoresis showing the topology of
pCOR pXL3179 produced in E. coli TEX2 pir42 (line B), in E. coli
TEX1pir42 (line C), in E. coli TEX1 (line D).
[0099] FIG. 21: Analysis of plasmid pXL3179 produced by
fermentation in E. coli TEX2pir42.
I--MATERIALS AND METHODS
A) Materials
[0100] 1) Culture Media
[0101] Complete LB, 2XTY and SOC media and minimal M9 medium
(Maniatis et al., 1989) were used. Agar media were obtained by
addition of 15 g of Difco agar. Furthermore, if necessary, these
media were supplemented with antibiotics (ampicillin or kanamycin)
at respective concentrations of 100 mg/l and 50 mg/l. The
chromogenic substrates X-Gal and X-Gluc were used at a
concentration of 40 mg/l.
[0102] 2) E. coli Strains, Plasmids and Bacteriophages
[0103] The E. coli strains, plasmids and bacteriophages used are
respectively identified in the examples below.
B) Methods
[0104] 1) Manipulation of the DNA
[0105] The isolation of bacterial DNA (plasmid and genomic) and
phage DNA (replicative form of M13), digestion with restriction
endonucleases, ligation of the DNA fragments, agarose gel
electrophoresis (in TBE buffer) and other standard techniques were
carried out according to the manufacturers' recommendations, for
the use of enzymes, or in accordance with the procedures described
in "Molecular Cloning: a Laboratory Manual" (Maniatis et al.,
1989).
[0106] The DNA size markers used during the electrophoreses are as
follows: 1 kb ladder (BRL) for the linear fragments and the
supercoiled DNA marker (Stratagene) for the undigested
plasmids.
[0107] Sequencing was carried out according to the Sanger technique
(Sanger et al., 1977) adapted to the automated method using
fluorescent dideoxynucleotides and Taq DNA polymerase (PRISM Ready
Reaction DyeDideoxy Terminator Cycle Sequencing Kit, Applied
Biosystems).
[0108] The oligodeoxynucleotides used (designated by "seq+no.", see
below) were synthesized on the "Applied Biosystems 394 DNA/RNA
Synthesizer" by the phosphoramidite method, using
.alpha.-cyanoethyl protecting groups (Sinha et al., 1984). After
synthesis, the protecting groups are removed by treatment with
ammonia. Two precipitations with butanol allow the oligonucleotide
to be purified and concentrated (Sawadogo et al., 1991).
[0109] Sequences of the Oligonucleotides Used for the PCR
Amplification:
TABLE-US-00002 5'-GACCAGTATTATTATCTTAATGAG-3' SEQ ID No. 3
5'-GTATTTAATGAAACCGTACCTCCC-3' SEQ ID No. 4
5'-CTCTTTTAATTGTCGATAAGCAAG-3' SEQ ID No. 5
5'-GCGACGTCACCGAGGCTGTAGCCG-3' SEQ ID No. 6
[0110] The PCR reactions (Saiki et al., 1985) were performed under
the following conditions, in a total volume of 100 .mu.l. The
reaction mixture comprises 150 ng of genomic DNA from the strain to
be studied, 1 .mu.g of each of the two oligonucleotide primers
(24-mer), 10 .mu.l of 10XPCR buffer, the composition of which is as
follows "500 mM KCl, 0.1% gelatin, 20 mM MgCl.sub.2, 100 mM
Tris-HCl pH 7.5", and 2.5 units of Taq DNA polymerase (Amplitaq
Perkin-Elmer). The PCR conditions, on the Perkin-Elmer Cetus DNA
Thermal Cycler machine are as follows: 2 min at 91.degree. C., 30
successive cycles of denaturation (1 min at 91.degree. C.),
hybridization (2 min at 42.degree. C.) and elongation (3 min at
72.degree. C.), and finally 5 min at 72.degree. C. The products
thus obtained, which are or are not digested with a restriction
enzyme, are analysed by agarose gel electrophoresis.
[0111] Analysis of the various plasmid species by DNA
topoisomerases was performed according to the following procedure:
the enzymes, purified in the laboratory, are incubated for 1 hour
at 37.degree. C. The reaction mixtures (total volume: 40 il) have
the following composition: 150 ng of plasmid, 300 ng of DNA
topoisomerase I or 150 ng of E. coli DNA gyrase, or 160 ng of S.
aureus DNA topoisomerase IV and 20 .mu.l of buffer specific for
each enzyme. The composition of these buffers is indicated
below:
[0112] for DNA topoisomerase I:
[0113] 50 mM Tris-HCl pH 7.7, 40 mM KCl, 1 mM DTT, 100 .mu.g/ml
BSA, 3 mM MgCl.sub.2, 1 mM EDTA;
[0114] for DNA topoisomerase IV:
[0115] 60 mM Tris-HCl pH 7.7, 6 mM MgCl.sub.2, 10 mM DTT, 100
.mu.g/ml BSA, 1.5 mM ATP, 350 mM potassium glutamate;
[0116] for DNA gyrase:
[0117] 50 mM Tris-HCl pH 7.7, 5 mM MgCl.sub.2, 1.5 mM ATP, 5 mM
DTT, 100 .mu.g/ml BSA, 20 mM KCl.
[0118] 2) Transformation of E. coli
[0119] This was performed routinely according to the TSB
(Transformation and Storage Buffer) method described by Chung and
Miller (1988). For a strain such as TG1 (Gibson et al., 1984), the
transformation efficiency obtained is about 10.sup.5-10.sup.6
transformants per .mu.g of pUC4K (Vieira and Messing; 1982). When a
higher transformation efficiency was necessary, the bacteria were
transformed by electroporation according to the procedure
recommended by the electroporator manufacturer (Biorad). This
method makes it possible to achieve efficiencies of from 10.sup.8
to 10.sup.10 transformants per .mu.g of pUC4K.
[0120] 3) Cellular Transfection Mediated by a Cationic
Lipofectant
[0121] The cells used are NIH 3T3 mouse fibroblasts seeded the day
before into 24-well plates, at a density of 50,000 cells per well.
The culture medium used is DMEM medium, containing 4.5 g/l of
glucose and supplemented with 10% foetal calf serum and 1% of
solutions of 200 mM glutamine and antibiotics (5.10.sup.3/ml
streptomycin, 5.10.sup.3 .mu.g/ml penicillin) (Gibco). The plasmid
DNA (1 .mu.g in 25 .mu.l of 9% O NaCl) is mixed, on a
volume-for-volume basis, with a suspension of lipofectant. Four
"lipofectant charges/DNA charges" ratios are tested: 0, 3, 6 and 9.
These ratios are calculated by considering that 1 .mu.g of plasmid
DNA carries 3.1 nmol of negative charges and that the lipofectant
contains 3 positive charges per molecule. After a contact time of
10 minutes to allow formation of the DNA/lipid complex, 50 .mu.l of
DNA-lipofectant mixture are introduced onto the cells in serum-free
culture medium (500 .mu.l). The cells were prerinsed twice with
this same medium. Inhibition of transfection by the serum is thus
avoided. After incubation (2 hours at 37.degree. C. in the CO.sub.2
incubator), 10% foetal calf serum is added to the medium. The cells
are then reincubated for 24 hours.
[0122] 4) Measurement of the Luciferase Activity of Eukaryotic
Cells
[0123] This is carried out 24 hours after the transfection.
Luciferase catalyses the oxidation of luciferin in the presence of
ATP, Mg.sup.2+ and O.sub.2, with concomitant production of a
photon. The total amount of light emitted, measured by a
luminometer, is proportional to the luciferase activity of the
sample. The reagents used are supplied by Promega (luciferase assay
system) and used according to the recommended procedure. After
lysis of the cells, the insoluble fraction from each extract is
eliminated by centrifugation. The assay is carried out on 5 .mu.l
of supernatant, which may or may not be diluted in the cell lysis
buffer.
[0124] 5) Measurement of the Protein Concentration in the Cell
Extracts
[0125] This is carried out according to the BCA method (Pierce)
using bicinchoninic acid (Wiechelman et al., 1988). The standard
BSA range is prepared in the lysis buffer (cf. III-B-4). The
samples to be assayed and those of the range are pretreated, on a
volume-for-volume basis, with 0.1 M iodoacetamide/0.1 M Tris
buffer, pH 8.2, for 1 hour at 37.degree. C. This treatment makes it
possible to prevent interference, during the assay, of the reducing
agent (DTT) present in the lysis buffer. The assay result is read
at 562 nm.
Example 1
Construction of XAC-1 pir and pir-116 host Strains by Homologous
Recombination
[0126] The strain used is the E. coli strain XAC-1 (Normanly et
al., 1980). The argE gene of this strain advantageously includes a
mutation of glutamine-53 (CAG) into the amber codon (TAG) (Meinnel
et al., 1992). The argE gene belongs to the argECBH divergent
operon and codes for an arginine biosynthesis enzyme,
N-acetylornithinase. XAC-1 cannot therefore synthesize arginine
and, consequently, grow in minimal medium. This auxotrophy will be
relieved if the strain harbours a plasmid which allows the
expression of a suppressor tRNA. It will thus be possible, by
culturing in minimal medium, to select bacteria which carry such a
plasmid. In order to allow the replication therein of plasmids
derived from R6K, it was necessary to introduce, by homologous
recombination, the pir gene into the genome of XAC-1. The pir gene
(wild-type or mutated) is introduced at the uidA locus by exchange
between the wild-type uidA gene and a copy interrupted by the pir
(or pir-116) gene. The uidA gene codes for .beta.-glucuronidase,
the enzyme for hydrolysis of .beta.-glucuronides. This gene may be
inactivated without any problem since it is not essential for
growth in standard synthetic media, in which .beta.-glucuronides
are not used. Furthermore, the .beta.-glucuronidase activity can be
monitored by means of a chromogenic substrate, X-Gluc, whose
hydrolysis releases a blue pigment.
[0127] 1) Construction of a Suicide Vector Carrying the Cassette
"Km.sup.R-uidA::pir (or pir-116)
[0128] We used a strategy involving a single bacterial host and
minimizing the modifications to the genome of the strain of
interest. The phage M13 mp10 (Messing et Vieira; 1982) was used as
suicide vector (Blum et al., 1989). An amber mutation in the gene
II, which is essential for replication, reduces the host spectrum
of this M13 to the strains, such as TG1 (supE), which produce an
amber suppressor tRNA; it will therefore not be able to replicate
in E. coli sup+ strains, such as XAC-1.
[0129] The 3.8 kb BamHI cassettes, containing the
kanamycin-resistance gene of Tn5 and _uidA::pir or pir-116, were
respectively purified from M13wm34 and 33 (Metcalf et al., 1994).
They were cloned into M13 mp10 linearized with BamHI. The
recombinant clones were selected by plating on LB+Km agar medium,
after electroporating the ligation mixtures into TG1. The
conformity of the clones obtained was shown by analysing the
restriction profile and by sequencing the region corresponding to
the pir-116 mutation.
[0130] 2) Introduction of the pir or pir-116 Genes into the Genome
of E. coli XAC-1 by Homologous Recombination
[0131] The strategy adopted and the various events involved are
presented in FIG. 3.
[0132] a) First Recombination Event
[0133] The XAC-1 strain was transformed by electroporation with 10,
100 or 2000 ng of each RF (mp10-_uidA::pir or pir-116). One-third
of each expression mixture was plated out on LB plates containing
kanamycin and incubated overnight at 37.degree. C. The
mp10-_uidA::pir or pir-116 phages cannot replicate in the strain
XAC-1 (sup+). The Km.sup.R marker can therefore only be maintained
by integration into the genome of the bacterium via a homologous
recombination with the wild-type copy of the gene uidA. The results
of the electroporations of XAC-1 are presented in Table 1. The
transformation efficiency obtained was 4.10.sup.9 transformants per
.mu.g of pUC4K.
TABLE-US-00003 TABLE 1 Number of colonies obtained with the amounts
of DNA transformed CONSTRUCT 10 ng 100 ng 2000 ng
M13mp10-_uidA::pir 1 41 146 M13mp10-_uidA::pir-116 0 16 124
[0134] Under the test conditions, the number of integrants
increases in a non-linear manner with the amount of DNA. Given the
transformation efficiency and the size of the RFs (11.7 kbp), it is
possible to have an approximate idea of the level of recombination.
By considering the point at 100 ng, a recombination frequency of
about 10.sup.-6 is obtained.
[0135] b) Second Recombination Event
[0136] The second recombination event will then be selected by the
resistance of the strains to deoxycholate (Doc.sup.R).
[0137] To do this, 5 integrants of each construct were cultured in
2XTY medium supplemented with 0.2% sodium deoxycholate. Two
distinct populations appeared. Certain clones give quite visible
cloudiness after about 8 hours at 37.degree. C. (two clones for the
pir construction and three for the pir-116 construction). The other
clones gave a dense culture only after one night at 37.degree. C.
They were virtually all Km.sup.S, as expected. For each of the
electroporants studied, 50 Km.sup.S descendants were streaked onto
LB medium supplemented with X-Gluc. After 48 hours at 37.degree.
C., the UidA.sup.+ clones were pale blue whereas those which had
undergone an allele replacement (case No. 1, FIG. 3) remained white
on this medium (UidA.sup.-). Table 2 summarizes the phenotype
analysis of the double recombinants obtained.
[0138] From 18 to 30% of the double recombinants underwent an
allele replacement.
TABLE-US-00004 TABLE 2 Number of Km.sup.S Percentage of UidA.sup.-
Strain among the Doc.sup.R among the Km.sup.S XAC-1 pir-2 50/50 18
XAC-1 pir-3 50/50 24 XAC-1 pir-4 50/50 34 XAC-1 pir-116-1 50/50 32
XAC pir-116-4 35/50 30
[0139] 3) Checking the Pir+ Character Nature of the Strains
Obtained by Recombination
[0140] In order to ensure the Pir+ character of the strains
obtained by double recombination, we transformed three clones of
each construct with pBW30 (Metcalf et al., 1994). The fact that
transformants were obtained for all the test strains made it
possible to show the functionality of the pir and pir-116 genes
which were integrated into the genome of XAC-1. Under the same
conditions, no transformant is obtained with the parental strain
XAC-1. We continued to study 2XAC-1pir clones (B and C) and
2XAC-1pir-116 clones (E and D).
[0141] 4) Checking, by PCR Amplification of the Strains Obtained by
Recombination
[0142] In order to confirm the allele replacement, we checked the
genomic regions on either side of the uidA locus by PCR
amplification. Each pair of oligonucleotides consists of an
oligonucleotide corresponding to an internal region of pir and a
second oligonucleotide corresponding to a region, close to
chromosomal uidA, but not within the fragment which served for the
recombination. The sequence of the latter oligonucleotide was
determined by means of the ECOUIDAA sequence from Genbank (access
number: M14641). We were thus able to verify the exact location of
the pir gene in the bacterial genome. The nature of the amplified
fragments, whose size is in accordance with that which might be
expected, was confirmed by digestion with MluI.
Example 2
Construction of Plasmid Vectors Derived from R6K Carrying the
Selection Marker sup Phe
[0143] Vectors were constructed containing ori .gamma. from R6K and
the kanamycin-resistance gene (pXL2666). The observation of pXL2666
multimers in the strain BW19610 (pir-116) 5 (Metcalf et al., 1993)
led us to study the effect of the cer fragment from ColE1 on this
phenomenon. We then introduced the expression cassette of the
phenylalanine suppressor tRNA (sup Phe) onto the vector ori
.gamma.-Km.sup.R-cer (pXL2730). This vector, pXL2760, serves as a
basis for the construction of vectors which can be used in gene
therapy.
[0144] 1) Construction and Analysis of Vectors Containing Ori
.gamma. from R6K and the Kanamycin-Resistance Gene
[0145] a) Constructs
[0146] In the first plasmid constructed, pXL2666, the
kanamycin-resistance gene originates from pUC4K (Vieira and
Messing; 1982) and the origin of replication, contained in a 417 bp
EcoRI-BamHI fragment, originates from the suicide vector pUT-T7pol
(Herrero et al., 1990) (FIG. 4). The transfer of pXL2666 into the
strains BW19094 and 19610 (Metcalf et al., 1994) made it possible
to show that the amount of plasmid is indeed increased in a pir-116
strain, when compared with the same plasmid in a pir strain.
However, the electrophoretic analysis of the undigested plasmids
shows that this increase goes hand in hand with the appearance of a
few multimeric forms. This phenomenon is quite probably associated
with intermolecular recombination between the multiple copies of
the plasmid. Thus, we constructed pXL2730 by cloning the cer
fragment of the natural E. coli plasmid, ColE1, which had been
shown to permit, in cis, the resolution of plasmid dimers (Summers
and Sherrat, 1984), into pXL2666. The fragment used corresponds to
a 382 bp HPAII fragment from ColE1 (Leung et al., 1985). It
contains a specific intermolecular recombination site; in order to
function, it involves only host proteins including the recombinases
XerC and XerD and the accessory factors ArgR and PepA (Stirling et
al., 1988, 1989; Colloms et al., 1990). In order to ensure that the
effects observed are indeed due to the cer fragment, we also
constructed the control plasmid pXL2754 in which the cer fragment
has a 165 bp deletion. This deletion was shown to abolish the
action of cer on the resolution of the multimers (Leung et al.,
1985). The various cloning steps leading to the construction of
these plasmids are presented in FIG. 4.
[0147] b) Quantitative and Qualitative Analysis of the Plasmid
Species
[0148] (i) Analysis by Agarose Gel Electrophoresis
[0149] Electrophoretic analysis of the different plasmids
constructed allowed the demonstration of various plasmid species,
which are variable according to the strains used. The size of the
undigested plasmids was evaluated relative to the supercoiled DNA
marker. In the pir strain (BW19094), the plasmids pXL2666, 2754 and
2730 are almost entirely in monomeric form. The bands above each
main band correspond to various slightly less supercoiled
topoisomers, as confirmed by the profile observed after the action
of DNA gyrase on pXL2730.
[0150] In the case of the pir-116 strain (BW19610), the profiles
are different: with the plasmids pXL2666 and 2754 different species
are observed ranging from the monomer to multimers (2, 3 or 4
units), the major form being the dimer. After digestion with EcoRI,
only the linear plasmid DNA is found; these plasmid species
correspond either to plasmid multimers or to various topoisomers.
However, since the size of the forms determined according to the
supercoiled DNA marker is a whole product of that of the monomer
plasmid, it is highly probable that they are multimers. The
formation of multimers is most probably attributable to the pir-116
mutation, although the two strains BW19094 and BW19610 are not
strictly isogenic (BW19610 is recA). The profile obtained with
pXL2730 is different: although multimeric forms are still visible,
the major form is the monomeric form. The cer fragment can thus
facilitate resolution of the plasmid multimers which we have
constructed, independently of recA, in BW19610.
[0151] (ii) Analysis after Treatment with DNA Topoisomerases
[0152] In order to disprove the theory that the forms observed in
the strains carrying the pir-116 allele are specific topoisomers,
each plasmid preparation was subjected to the action of DNA
topoisomerases. The activities of the various enzymes under the
experimental conditions are as follows: relaxing of DNA for E. coli
DNA topoisomerase I, negative supercoiling of relaxed DNA for E.
coli DNA gyrase, and disentanglement of interlaced DNAs and
relaxation of supercoiled DNA by S. aureus DNA topoisomerase IV.
The action of DNA topoisomerase IV made it possible to show that
the high-molecular-weight plasmid forms did not result from the
entanglement of several plasmid molecules; in this case, they would
then have been converted into the monomeric species. The
functionality of the enzyme was, of course, checked on a
preparation of kinetoplast DNA, composed of entangled DNA molecules
(not shown). The relaxation activity is also visible since species
are obtained which migrate less than in the untreated controls. The
action of DNA gyrase made it possible to convert the slightly
relaxed topoisomers into the more supercoiled species extracted
from the bacterium (monomer or dimer mainly). Furthermore, it made
it possible to verify that the DNAs prepared are mainly in
supercoiled form. The samples thus treated allow the above results
to be confirmed as regards the major species for each construct.
DNA topoisomerase I did indeed relax DNA, but only partially. This
could be due to the fact that the plasmids studied contain only a
few single-stranded regions, to which this enzyme preferably binds
(Roca, 1995).
[0153] 2) Introduction of the Selection Marker Sup Phe into
pXL2730
[0154] We used the expression cassette of the synthetic suppressor
tRNA gene (Phe) (Kleina et al., 1990). This introduces a
phenylalanine into the growing polypeptide chain in response to a
TAG codon. Furthermore, it allows the production in XAC-1 of an
ArgE protein which is sufficiently active to allow good growth in
arginine-deficient medium. sup Phe is expressed constitutively on
the plasmid pCT-2-F (Normanly et al., 1986), from a synthetic
promoter derived from the promoter sequence, Plpp, of the E. coli
lpp gene. Downstream of this gene, transcription is stopped by the
synthetic terminator, T.sub.rrnC, of the E. coli operon rrnC
(Normanly et al., 1986). The various cloning steps are indicated in
FIG. 5.
[0155] The various subclonings were performed in XAC-1. The
functionality of the suppressor tRNA expression cassette is thus
checked by means of the .alpha.-galactosidase activity of this
strain, which only exists if there is suppression of the amber
codon of the gene lacZ.sub.u118am. The final step consists of the
introduction of the sup Phe expression cassette into pXL2730. The
results obtained with the cer fragment (B-1-b) led us to select
this plasmid rather than pXL2666. We retained the
kanamycin-resistance gene for ease of subsequent cloning, in
particular in order to have available additional screening during
the final cloning (loss of Km.sup.R).\
Example 3
Validation of the Plasmid Vector for Applications in Gene Therapy
by Transfection of Mouse Fibroblasts
[0156] 1) Construction of the Reporter Vector pXL2774
[0157] In order to test the validity for gene therapy of the system
for producing plasmid DNA, we introduced a reporter gene, which can
be used in eukaryotic cells, into pXL2760. We used the gene luc
coding for Photinus pyralis luciferase since the bioluminescence
measurement test is very sensitive and is linear over a large
range, and the background noise due to the endogenous activity of
eukaryotic cells is very low. The luc gene is controlled by
promoter-enhancer sequences of a human cytomegalovirus early gene
(CMV promoter) which allows a high level of expression. There is an
untranslated region at the 3' end of luc, originating from the
virus SV40, which contains the polyadenylation signal (poly(A)+).
After intermediate cloning which allows the number of available
restriction sites to be increased, the "CMV promoter-luc-poly(A)+"
cassette is introduced into the minimal vector ori .gamma.-cer-sup
Phe (pXL2760) in place of the Km.sup.R marker. The resulting
plasmid has been named pXL2774. FIG. 5 collates the various cloning
steps. The ligation mixtures were transformed into XAC-1pir-116 by
electroporation. Incubation allowing the bacteria to express
selection markers is carried out in rich medium (SOC medium); it
was thus necessary to wash the cells twice with M9 medium before
plating out. This made it possible to remove the residual medium
which would have resulted in culture background noise on minimal
medium.
[0158] The medium chosen to plate out the electroporated cells is
M9 minimal medium, which makes it possible to select bacteria
expressing a suppressor tRNA and thus the presence of our plasmids.
The addition of X-Gal makes it possible, by means of the blue
colouration, to visualize the expression of the suppressor tRNA.
The dishes are analysed after about 20 hours at 37.degree. C. The
absence of colonies on the DNA-free control assures us that the
selection is correct, even with dense seedings. All the clones
examined by restriction (8) do indeed carry a plasmid,
corresponding to the expected profile. The plasmid thus
constructed, pXL2774, was prepared from a clone cultured in one
litre of liquid M9 medium (about 18 hours at 37.degree. C.), by a
technique involving, inter alia, an ion-exchange (Promega kit,
MegaPreps). The amount of DNA collected is 2 mg.
[0159] 2) Analysis of the Reporter Vector pXL2774 Transfected into
Mammalian Cells.
[0160] The capacity of pXL2774 to transfect eukaryotic cells and to
allow the expression of luciferase is evaluated by transfection
into NIH 3T3 mouse fibroblasts. The vector chosen as reference is
the plasmid pXL2622 (this is the plasmid pGL2 from Promega whose
SV40 promoter has been replaced by the CMV promoter) which carries
the same luciferase expression cassette as pXL2774, but on a
different replicon. This is a 6.2 kb ColE1 derivative which carries
the ampicillin-resistance gene. This plasmid serves as a control.
The luciferase activities (expressed as RLU, or relative
luminescence units) are indicated in Table 3.
[0161] The best results were obtained with a "lipofectant
charges/DNA charges" ratio of 6; under these conditions, pXL2622
and 2774 appear to be equivalent.
TABLE-US-00005 TABLE 3 pXL2622 pXL2774 RLU/.mu.g of Coefficient
RLU/.mu.g of Coefficient Charge proteins and of variation proteins
and of variation ratios per well Average (%) per well Average (%) 0
0.0 not 0.0 not 0.0 detectable 0.0 detectable 0.0 0.0 3 9.9
10.sup.6 7.6 10.sup.6 22 3.3 10.sup.6 2.9 10.sup.6 13 6.2 10.sup.6
2.9 10.sup.6 6.6 10.sup.6 2.4 10.sup.6 6 1.2 10.sup.7 1.5 10.sup.7
19 9.4 10.sup.6 1.0 10.sup.7 7 1.5 10.sup.7 9.9 10.sup.6 1.9
10.sup.7 1.1 10.sup.7 9 9.5 10.sup.6 1.0 10.sup.7 26 1.1 10.sup.7
6.4 10.sup.6 13 7.5 10.sup.6 8.3 10.sup.6 1.4 10.sup.7 8.5
10.sup.6
Example 4
Verification of the Suicide Vector Nature in E. coli of the pCOR
Plasmids
[0162] The non-replicative nature of the pCOR-type plasmids derived
from R6K was verified by an electroporation experiment in JM109 E.
coli (Yanisch-Perron et al., 1985) of the plasmids pUC4K (ori
ColE1-KmR, (Vieira and Messing, 1982)) and pXL2730 (ori gamma from
R6K-KmR, see Example 2). The electroporator used is the Biorad Gene
Pulser and the electrocompetent JM109 cells are prepared and used
according to the manufacturer's procedure (Bacterial
electro-transformation and pulse controller instruction manual.
catalog number 165-2098).
[0163] The electrotransformed cells were plated out on LB medium
supplemented with kanamycin (50 mg/l) and incubated overnight at
37.degree. C. The results obtained are presented below.
Results
TABLE-US-00006 [0164] Efficacy (number of Amount transformed Number
of transformants/ Plasmid (ng) transformants ng of plasmid) pUC4K
0.01 >>2000 >2105 pXL2730 5 0 0
[0165] These results show that there is a minimum of 5 log of
difference between the efficacy of transformation of a ColEI
derivative (pUC4K) and that of an R6K derivative (pXL2730) in a
strain which does not express the pir gene. In a pir+ strain such
as XAC-1pir-116, the electrotransformation efficacy of R6K-derived
plasmids conventionally reaches or exceeds the 108
transformants/.mu.g of plasmid.
Example 5
Production of Plasmid DNA by High-Density Culturing of the E. Coli
Strain XAC-1pir-116 (pXL2774): Fermentation Process
[0166] 5.1. Strains:
[0167] Production in XAC-1pir-116 E. coli (Example 1) of a minimal
plasmid, pXL2774; this plasmid comprises the following elements:
ori R6K-cer-tRNAamsupPhe and an expression cassette of the luc
reporter gene under the control of the CMV promoter (Example 3). A
high-productivity process for the production of plasmids of this
type was developed.
[0168] 5.2. Culturing Media and Conditions:
[0169] a) Growth Medium:
[0170] Composition of the medium defined used for the inoculum
cultures (g/l): Na.sub.2HPO.sub.4 6, KH.sub.2PO.sub.4 3, NaCl 0.5,
NH.sub.4Cl 1, NH.sub.4H.sub.2PO.sub.4 3, glucose 5,
MgSO.sub.4.7H.sub.20 0.24, CaCl.sub.2.2H.sub.2O 0.015, thiamine HCl
0.010 [0171] Composition of the complex medium used for the
fed-batch cultures (g/l): KH.sub.2PO.sub.4 8, K.sub.2HPO.sub.4 6.3,
Na.sub.2HPO.sub.4 1.7, (NH.sub.4).sub.2SO.sub.4 0.74, NH.sub.4Cl
0.12, yeast extract 3, glucose 2, MgSO.sub.4.7H.sub.20 2.4 g/l,
CaCl.sub.2.2H.sub.2O 0.015, thiamine 0.010, solution of salts (Fe,
Mn, Co, Zn, Mo, Cu, B, Al).
[0172] Composition of the medium defined for the cultures in
fed-batch medium identical to the complex medium but the yeast
extract is replaced by 2.5 g/l of NH.sub.4Cl.
[0173] b) Conditions of Fed-Batch Culturing:
[0174] Studies in 2-litre fermenters (Setric France) containing 1 l
of medium were carried out in order to define the optimum
conditions for growing and producing plasmid DNA. The fermenter was
inoculated with 80 ml of an inoculum culture arrived at the start
of the stationary phase of growth.
[0175] During the fermentation, the pH was controlled and adjusted
automatically between 6.9 and 7.0 with 10% (w/v) aqueous ammonia;
the temperature is maintained at 37.degree. C.; the aeration was
set at 75 l/h ((1.1 vvm) at a pressure of 0.2 bar and the dissolved
oxygen was adjusted to (40% of air saturation by retroaction on the
stirring rate and, if necessary, by enrichment with pure
oxygen.
[0176] All the parameters (pH, temperature, stirring, OD, O.sub.2
and CO.sub.2 in the effluent gases) were collected and calculated
in line via an HP3852 interface connected to a Hewlett-Packard
9000.
[0177] The base composition of the supply medium is as follows: 50%
carbon source, 0.7% magnesium sulphate, 0.02% thiamine; for the
complex medium, yeast extract was added to a concentration
preferably of between 5 and 10%.
[0178] In order to adapt the culture conditions to 800-litre
fermenters, production sequences composed of two successive
inoculum cultures were carried out, on a laboratory scale: inoculum
I in an agitated conical flask and inoculum II in a 2-litre
fermenter (batch culturing), followed by fed-batch production
culturing, in a 7-litre fermenter.
[0179] 5.3. Results
[0180] Various culture conditions were studied in complex medium,
in defined medium, and at various growth rates. In all cases, after
initial batch culturing of the bacterial strain and consumption of
the carbon source, the supply medium was added to the fermenter by
means of a peristaltic pump coupled to a pre-programmed addition
profile. This profile was deduced from previous experiments in
which the supply rate had been controlled either by the level of
dissolved oxygen or by a constant growth rate.
[0181] Furthermore, in order to extrapolate without difficulty the
2-litre fermentation condition to an 800 l fermenter without
overoxygenation of the medium, the maximum oxygen demand at the end
of the culturing was set at 2.5-3 mM/min. For this, the growth rate
of the microorganism was reduced, if necessary, by varying the
supply rate of the complementary charge.
[0182] As seen in Table 4, very good results were obtained both in
complex medium and in defined medium, both on the laboratory scale
and on the 800-litre fermenter scale; furthermore, the plasmid DNA
growth and production kinetics are entirely comparable (cf. FIGS. 6
and 7).
TABLE-US-00007 TABLE 4 Defined Complex medium medium 2 or 7 l 800 l
2 l fermenter fermenter fermenter Duration of 40 39 48 fermentation
(hours) .mu.h-l 0.130 0.132 0.124 OD (600 nm) 114 100 94 X g/l 44
37 30 Plasmid DNA 115 100 100 (mg/l medium) Plasmid DNA 2.6 2.7 3.3
(mg/gX) X = Biomass (weight of dry cells)
[0183] From the overall results it emerges that: [0184] changing
the scale of the fermentor from 2 litres to 800 litres can be
carried out without any problem, [0185] the oxygen consumed is
strongly correlated to the biomass produced (1.08 g O.sub.2/g of
biomass produced), [0186] the plasmid is stable for at least 50
generations without selection pressure, [0187] a high biomass,
greater than 40 g of dry cells/litre, can be obtained in complex
medium, [0188] the plasmid DNA production reaches 100 mg of
supercoiled DNA/l of medium, [0189] there is very good correlation
between the DNA production and the biomass: the production can be
estimated to (1 mg of plasmid DNA/OD unit, or alternatively (2.7 mg
of plasmid DNA/g of biomass, irrespective of the duration of
fermentation, [0190] the use of a defined medium also makes it
possible to achieve a high biomass (30 g of dry cells/l) and high
plasmid DNA production (100 mg/l), without any loss of
productivity.
Example 6
Transfer of pXL2774 into Animal Cells, In Vitro and In Vivo
[0191] 6.1. In Vitro Transfer of pXL2774 into Animal Cells
[0192] The capacity of the minimal plasmid pXL2774 to transfect
various cell lines was tested in vitro, on cells of both human
origin and murine origin. The pXL2784 plasmid was used as control.
It contains the same eukaryotic expression cassette (CMV
promoter-luciferase-polyA) as pXL2774, but this is a 6.4 kb ColEI
derivative which comprises the gene for imparting kanamycin
resistance in E. coli.
[0193] The cells tested are the following:
TABLE-US-00008 Atcc ref./ Cells Type literature ref. 3LL Mouse
pulmonary carcinoma NIH 3T3 Mouse embryo fibroblasts CCL92 293
Human embryo renal cells transformed CRL1573 with type-5 adenovirus
HeLa Human carcinoma from the neck of the CCL2 womb Caco-2 Human
colon adenocarcinoma HTB37 H460 Human lung carcinoma with no small
HTB177 cells ECV 304 Human umbilical cord endothelial cells
Takahashi et al., 1990
[0194] The transfection conditions were as follows:
[0195] D-1: Inoculation of the cells at a density of 100,000 cells
per 2 cm.sup.2 well (24-well plate) in DMEM medium (Dulbecco's
modified Eagle Medium) supplemented with 10% foetal calf serum
(FCS).
[0196] D-3: Transfection of the cells, by 10 .mu.l of a
transfection solution containing: 0.5 .mu.g of DNA, 150 mM NaCl, 5%
glucose and 3 nmol of RPR120 535 lipofectant per .mu.g of DNA, in
250 .mu.l of culture medium, which is or is not supplemented with
10% FCS. After incubation for 2 hours, the medium is replaced by
500 .mu.l of DMEM medium supplemented with 10% FCS.
[0197] D-4: Renewal of the culture medium
[0198] D-5: Washing of the cells with PBS, followed by lysis with
100 .mu.l of Promega lysis buffer (Promega Cell Lysis Buffer E153
A). Assay of the luciferase activity is carried out in a Lumat LB
9501 luminometer (Berthold) on 10 .mu.l of lysate, with a 10-second
duration of integration. The reactant used is that from Promega
(Promega Luciferase Assay Substrate). The results, collated in the
following tables 5-8, are expressed in RLU (Relative Lights Units)
for 10 .mu.l of lysate (average of measurement on 4 wells). The
coefficients of variation (CV) are also given.
[0199] The results of transfections in the absence of serum are
presented below.
TABLE-US-00009 CELL TYPES NIH 3T3 3LL 293 pXL2774 37 763 380 559
270 1 884 200 RLU 16 25 73 CV pXL2784 113 764 1 723 546 RLU 24 101
CV CELL TYPES HeLa CaCo2 H460 ECV304 pXL2774 11 000 000 1 108 422 1
459 501 36 450 15 14 5 23 pXL2784 557 930 93 610 7 563 168 795 87
40 11 40
[0200] The results of transfections in the presence of serum (10%)
are presented below:
TABLE-US-00010 CELL TYPES NIH 3T3 3LL 293 pXL2774 50 612 590 566
377 992 500 12 18 59 PXL2784 12 693 780 436 704 2 300 000 38 12 47
CELL TYPES HeLa H460 ECV304 pXL2774 9 490 000 857 385 18 021 25 16
30 PXL2784 1 508 480 433 023 32 074 23 27 47
[0201] These results reveal the capacity of pXL2774 to transfect
effectively, in vitro, various cell types of both murine and human
origin. The expression of the luc reporter gene makes it possible
to show that its transfection efficacy is at least as good as that
of a "standard" plasmid, derived from ColE1, which carries the same
expression cassette of luciferase.
[0202] 6.2. In Vivo Transfer, in Animals (Mice), of pXL2774
[0203] a) Model 1: Naked DNA in Mouse Cranial Tibial Muscle
[0204] Naked plasmid DNA, dissolved in "5% glucose, 150 mM NaCl" is
injected into the cranial tibial muscle of OF1 mice. The muscles
are removed 7 days after injection, chopped up, homogenized in 750
.mu.l of lysis buffer (Promega Cell Lysis Buffer E153A) and then
centrifuged at 20,000.times.g for 10 minutes.
[0205] Assay of the luciferase activity is carried out on 10 .mu.l
of supernatant after addition of 50 .mu.l of reagent (Promega
Luciferase Assay Substrate). The reading is carried out on a Lumat
LB9501 luminometer (Berthold) with a 10-second duration of
integration.
[0206] The results are presented in the table below.
TABLE-US-00011 Plasmid pXL2784 pXL2774 pXL2784 pXL2774 Number of 8
8 10 10 muscles: Volume injected 30 30 33 33 (il): .mu.g of 19 13.5
10 6.25 DNA/muscle RLU (for 10 .mu.l) Average 80 922 471 733 35329
30569 Standard deviation 104 573 402 602 37041 35774
[0207] These results show that a conditional replication plasmid
such as pXL2774 is indeed capable of transfecting mouse muscle
cells in vivo and of doing so with comparable, or even superior,
efficacy to that of a "standard" plasmid, derived from ColE1, which
carries the same expression cassette of the luciferase gene.
[0208] b) Model 2: 3T3 HER2 Tumour Model
[0209] The model is as follows: [0210] Swiss/nude adult female type
mice [0211] Experimental tumours induced after injection of 107 3T3
HER2 cells subcutaneously into the flank. [0212] The transfection
mixture is injected 7 days after injection of the cells.
[0213] Solutions injected: The DNA is first dissolved in the
buffer. After addition of all the products, the mixture contains,
besides the DNA, NaCl (150 mM) and 5% D-glucose in water or 5 mM
HEPES buffer. [0214] Two days after the injection, the tumour
tissue is removed, weighed and then chopped up and homogenized in
750 il of lysis buffer (Promega Cell Lysis Buffer E153 A). After
centrifugation (20,000.times.g for 10 minutes), 10 .mu.l of
supernatant are removed and allow the luciferase activity to be
evaluated. This activity is determined by measuring the total light
emission obtained after mixing with 50 .mu.l of reagent (Promega
Luciferase Assay Substrate) in a Lumat LB 9501 luminometer
(Berthold) with a 10-second duration of integration.
[0215] The resulting activity is expressed in RLU (Relative Light
Units) estimated in the entire tumour lysis supernatant.
TABLE-US-00012 RESULTS Plasmid Buffer [DNA] RLU/tumour results H20
or final in standard HEPES reference .mu.g/tumour inj. sol. average
deviation +/n HEPES pXL2784 10 0.5 .mu.g/.mu.l 744 150 682 434 6/6
pXL2774 10 0.5 .mu.g/.mu.l 1 016 380 1 322 500 5/6 H2O pXL2784 24
0.6 .mu.g/.mu.l 2 906 073 1 745 857 8/8 pXL2774 16.8 0.4
.mu.g/.mu.l 4 292 043 4 995 187 6/6 H2O pXL2784 7.5 0.3 .mu.g/.mu.l
702 554 552 207 6/7 pXL2774 5 0.2 .mu.g/.mu.l 3 413 430 4 000 875
6/6
[0216] These results show that a conditional replication plasmid,
such as pXL2774, is indeed capable of transfecting mouse tumour
cells in vivo and of doing so with an efficacy at least comparable
to that of a "standard" plasmid, derived from ColE1, which carries
the same expression cassette of the luciferase gene.
[0217] These various experiments made it possible to demonstrate
that the conditional replication plasmids, and more particularly
pXL2774, did indeed have animal cell transfection characteristics
that are essential for use in gene therapy. More precisely, the
following were shown:
[0218] 1) the capacity of pXL2774 to transfect efficiently, in
vitro, various cell types, of human or murine origin;
[0219] 2) the capacity of pXL2774 to transfect, in vivo, mouse
muscle;
[0220] 3) the capacity of pXL2774 to transfect, in vivo, tumour
cells implanted into mice.
[0221] The electrotransformation, fermentation and transfection
experiments thus made it possible to validate conditional
replication plasmids as vectors which can be used in gene therapy
by showing
[0222] i) that they did not replicate detectably in an E. coli
strain which does not express the pir gene (conditional origin of
replication)
[0223] ii) that they could be produced on a scale compatible with
industrial production, in a medium which can be totally defined and
which does not contain antibiotics;
[0224] iii) that these plasmids could transfect, in vitro and
especially in vivo, mammalian cells.
Example 7
In Vitro Production of Recombinant Proteins
[0225] 7.1. Construction of the Expression Vector
[0226] In order to show the feasibility of such an approach, we
constructed an expression vector according to the criteria
described above (Examples 2 and 3). This vector, pXL3056,
contains:
[0227] 1) the bacterial part which comprises the conditional origin
of replication (ori gamma), the cer fragment of ColE1 and the gene
which ensures selection in bacteria (sup)
[0228] 2) the expression cassette, based on the system described by
Studier (Studier et al., 1990), comprises the promoter of gene 10
of bacteriophage T7, the lacO operator, the gene coding for aFGF
154 (acidic Fibroblast Growth factor, form containing 154 amino
acids) (Jaye et al., 1986), the TF terminator of bacteriophage T7.
This expression cassette is identical to the one present on the
pXL2434 plasmid described in application WO 96/08572.
[0229] The construction of pXL3056 is presented in FIG. 8. The
EcoRI-BglII fragment of pXL2434 (1.1 kb) containing the aFGF
expression cassette is cloned in the pXL2979 conditional
replication vector (1.1 kb purified fragment) at the BglII and
EcoRI sites in order to generate pXL3056.
[0230] pXL2979 results from the ligation of 3 fragments: i)
AccI-XbaI fragment of pXL2730 (0.8 kb, which provides ori gamma and
cer), ii) NarI-SalI fragment of pXL2755 (0.18 kb, which provides
the sup Phe gene), iii) SalI-SpeI fragment of pXL2660 (1.5 kb,
which provides the kanamycin-resistance gene).
[0231] pXL2660 results from the cloning of the 1.2 kb PstI fragment
of pUC4K (Vieira and Messing, 1982) in pMTL22 (Chambers et al.,
1988) linearized with PstI.
[0232] 7.2. Production of the Expression Strain
[0233] The plasmid pXL3056 is introduced by transformation into the
XAC-1pir-116 strain. The resulting strain is then transformed by
the plasmid PT7pol23 (Mertens et al., 1995), at 30.degree. C. In
order to express the gene of interest under control of the T7
promoter, the bacterium must contain, in its genome, on a plasmid
or a bacteriophage, a cassette to allow the expression of the RNA
polymerase of bacteriophage T7. In the example described, we used
the plasmid PT7pol23, which is compatible with R6K derivatives such
as pXL3056, and which allows the temperature-inducible expression
of bacteriophage T7 RNA polymerase. However, it can also be
envisaged to lysogenize the XAC-1pir-116 strain with lambda DE3
(Studier et al., 1990) in order to conserve only one plasmid and to
induce the production of T7 RNA polymerase by IPTG rather than by
temperature.
[0234] 7.3. Expression of aFGF
[0235] The XAC-1pir-116 strain (pXL3056+PT7pol23) is cultured at
30.degree. C., in M9 minimum medium supplemented with 0.2% of
casamino acids (DIFCO) and kanamycin (25 .mu.g/ml), up to an
optical density at 600 nm of 0.6-1. Half of the culture is then
placed at 42.degree. C. (induction of the T7 RNA polymerase), while
the other half remains at 30.degree. C. (negative control). The
same experiment is carried out with the XAC-1pir-116
(pXL3056+pUC4K) strain which constitutes a control for the
expression of aFGF in the absence of T7 RNA polymerase.
[0236] The results obtained are presented in FIG. 9. They show that
the production of aFGF is comparable or superior to that observed
with BL21(DE3)(pXL2434) (WO 96/08572), which clearly shows the
potential of conditional replication plasmids for the expression of
recombinant proteins in vitro, especially in E. coli.
Example 8
Construction of a pCOR Vector which Expresses a Wild-Type or Hybrid
P53 Protein
[0237] This example describes the construction of conditional
replication vectors according to the invention containing a nucleic
acid which codes for a p53 protein. These vectors can be used to
restore a p53-type activity in deficient (mutated, deleted) cells
such as, in particular, tumour cells.
The eukaryotic expression cassette contains the following
elements:
[0238] 1) CMV "immediate early" promoter (positions -522 to +72)
followed by the leader sequence of the thymidine kinase gene of
type I herpes simplex virus (position -60 to +1 of the gene, with
reference to the sequence in the article by McKnight, S..dagger.L.
(1980) Nucleic Acids Res. 8:5949-5964);
[0239] 2) a nucleic acid which codes for wild-type p53 protein or
for a p53 variant, as described in application PCT/FR 96/01111
(V325K variant=V325 with a Kozak sequence with ATG);
[0240] 3) The polyA Polyadenylation Sequence of SV40.
[0241] These elements were placed in the form of a fragment
AscI-XbaI on the pCOR vector pXL2988 between the sites BssHII and
SpeI. pXL2988 is identical to pXL2979 (Example 7.1.) apart from the
presence of an additional element, a sequence capable of forming a
DNA triple helix composed of 17 times the trinucleotide GAA, placed
alongside the gamma origin of replication.
[0242] The resulting plasmids are named pXL3029 and 3030 (FIG.
10).
[0243] The functionality of these constructions was verified in
vitro on p53-SAOS2 cells in culture by measuring the
transcriptional-activator activity of p53 or p53superWT.
Example 9
Construction of TEX 1 (XAC1 pir116, endA.sup.-, traD.sup.-)
[0244] The E. coli XAC-1pir116 contains an F' episome, a circular
DNA molecule of approximately 100 kb, that carries
proB.sup.+lacI.sub.373lacZ.sub.u118am. Many male E. coli laboratory
strains carry a traD36 mutation on their episome, but no mutation
affecting F' transfer ability has been described for XAC-1. traD is
at the 5' end of one of the tra (transfer) operons and encodes a
membrane protein that is directly involved in DNA transfer and DNA
metabolism (Frost et al., BBRC, 1994, 58:162-210). A 2 kb central
fragment from traD, comprising 92% of the gene, was replaced with
the 2 kb omega element (Genbank accession number M60473) from
pHP45.OMEGA. (Prentki and Krisch, 1984, Gene, 29:303-313) by
homologous recombination in XAC-1 pir116 endA.sup.-. The omega
element contains the aadA antibiotic resistance gene flanked by
short inverted repeats. aadA encodes aminoglycoside-3
adenyltransferase and confers resistance to streptomycin and
spectinomycin (Sp.sup.R). The omega fragment was used because it
prematurely terminates RNA and protein synthesis leading to the
inactivation of the whole traD operon. This new pCOR strain XAC-1
pir-116 endA- traD::SpR was designated TEX1. Transfer of any
resident plasmids, either pCOR or pUC was undetectable when the
donor was TEX1.
[0245] The new pCOR host strain TEX1 was assessed in fermentation
experiments. Complex media containing yeast extract were used for
fed-batch fermentation with XAC-1pir 116. pCOR stability (more than
50 generations) makes it possible to use a non-selective media.
Under these conditions, XAC-1pir116 produced more than 40 g/l dry
cell weight and 100 mg/l of pCOR pXL2774 were obtained from 2-liter
fermentors. pCOR copy number was estimated at 400-500 copies per
cell and the rate of plasmid DNA synthesis was constant throughout
fermentation. These results were extrapolated to an 800-liter
fermentor suitable for manufacturing. The fermentation was also
performed in the absence of yeast extract or any raw material from
animal origin. Similar results (30 g/l dry cell weight and 100 mg/l
of plasmid DNA) were obtained using a defined medium in 2-liter
cultures with no loss of productivity.
Example 10
Construction of XAC-1pir-116 pir42 Host Strains by Homologous
Recombination
[0246] 1) Construction of a Suicide Vector Carrying the Cassette
"KmR-uidA:pir116;pir-42"
[0247] The Km.sup.R-uidA::pir-116 cassette from M13wm33 as
described in Example 1 (Metcalf W. et al. Gene, 1994, 138(1-2): p.
1-7), was modified by site-directed mutagenesis using PCR
(QuickChange site-directed mutagenesis kit, Stratagene, La Jolla,
Calif.) in order to introduce the pir42 mutation into the pir116
gene. The different cloning/mutagenesis steps are described in FIG.
13
[0248] The oligonucleotides used for mutagenesis contained the
pir42 mutation along with a silent mutation that created a ClaI
site to easily indicate the processing of pir42 by restriction
analysis when needed.
[0249] The sense and antisense oligonucleotides used are as
follows:
[0250] Sense oligonucleotide number 11076 9 (SEQ ID NO: 7)
TABLE-US-00013 5'-G TAT ATG GCG CTT GCT CTC ATC GAT AGC AAA GAA
pir42 ClaI CC-3'
Antisense oligonucleotide number 11077 (SEQ ID NO: 8)
TABLE-US-00014 5'-GG TTC TTT GCT ATC GAT GAG AGC AAG CGC CAT ATA
ClaI pir42 C-3'
[0251] The technique used to replace pir116 by pir116-pir42 in the
genome of E. coli pCOR host TEX1 was based on that of Blum et al.
(J. Bacteriol. 1989, 171, pp 538-46). The recombinant bacteriophage
pXL3723 shown in FIG. 13 is a suicide vector in all non-suppressor
E. coli strains, because it has a non-sense mutation in gene II
encoding M13 nickase that prevents viral genome replication.
[0252] Double recombination was performed as described for the
construction of XAC-1pir116 (Example 1, point 2). Clones that had
undergone double homologous recombination events were screened by
PCR to test for the presence of the pir42 mutation in the genome of
TEX1. Genomic DNA isolated from double recombination candidates was
used as a template for PCR. Secondly, sequencing was done on each
unique amplified fragment, all of which were of the expected size.
The PCR fragments are shown in FIG. 14.
[0253] The PCR primers were the following:
TABLE-US-00015 Primer 11088 (SEQ ID NO: 9):
5'-GAGATCGCTGATGGTATCGG-3' Primer 11089 (SEQ ID NO: 10):
5'-TCTACACCACGCCGAACACC-3'
[0254] This analysis showed that one out of the six double
recombinants tested had undergone the allele exchange. This new
strain was named TEX1pir42 and further evaluated for its ability to
replicate or not pCOR plasmids compared to the parental strain
TEX1.
[0255] 2) Evaluation of TEX1pir42
[0256] pCOR plasmids were transformed in parallel into TEX1 and
TEX1pir42 and grown overnight in 2 ml of selective M9 medium. Then,
the plasmid DNA was extracted with the Wizard SV plus minipreps kit
(Promega) in order to evaluate the relative plasmid copy number and
topology of the pCOR plasmids in both strains.
[0257] A 2-fold increase in copy number was obtained reproducibly
in TEX1pir42 transformed with the pCOR plasmid pXL3516 (2.56 kb).
To further characterize TEX1pir42, the copy number and topology of
pCOR plasmids such as pXL3179 and pXL2774 were evaluated by agarose
gel electrophoresis analysis after small scale purification of
plasmid DNA (4 to 6 clones/strain). Copy number was evaluated on
plasmids linearized with EcoRI restriction enzyme. A topology test
was run on non-digested plasmids, in the absence of ethidium
bromide. The resulting agarose gel is displayed in FIG. 15, and
clearly shows a higher plasmid copy number when the plasmid pXL3179
was produced in TEX1pir42, than when produced in TEX1 strain. FIG.
15 also displays the topology of the plasmid pXL3179, and shows
that an increase in plasmid copy number, which are essentially in
the form of monomers, with few plasmids in the form of multimers.
The results obtained with these pCOR plasmids are also summarized
in Table 5. Relative copy number was calculated in comparison with
the same plasmid in TEX1. A 2-3 fold increase in plasmid copy
number was observed with plasmids pXL3179 and 2774 produced in
TEX1pir42.
TABLE-US-00016 TABLE 5 Replication and copy number of pCOR plasmids
produced in TEX1pir42 RELATIVE COPY PLASMIDS SIZE (kb) NUMBER*
pXL3179 2.4 x3 pXL2774 4.5 x2 *copy number was compared to the same
plasmid in TEX1.
Example 11
Comparative Experiments: Construction of TEX1cop21 (XAC-1endA-
traD-pir116cop21)
[0258] 1) Construction of TEX 1 cop21
[0259] The TEX1cop21 strain was constructed similarly as that
described in Example 10 for TEX1pir42. The following
oligonucleotides used to introduced cop21 into the pir116 gene by
directed mutagenesis were as follows:
Sense oligonucleotides: 11153 (SEQ ID NO: 11)
TABLE-US-00017 5'-CG CAA TTG TTA ACG TCC AGC TTA CGC TTA AGT cop21
AGC C-3'
Antisense oligonucleotide: 11154 (SEQ ID NO: 12)
TABLE-US-00018 5'-G GCT ACT TAA GCG TAA GCT GGA CGT TAA CAA TTG
CG-3'
[0260] The cop21 mutation was introduced as a TCC Serine codon
instead of the TCA Serine codon to eliminate a HindIII restriction
site close to the mutation.
[0261] The template used for directed mutagenesis was pXL3395 (See
FIG. 13). The resultant plasmid named pXL3432 was used to construct
the suicide M13 vector in a similar way as what is described for
pir42 in FIG. 13. The suicide vector pXL3749 is presented in FIG.
16.
[0262] The E. coli clones obtained after homologous recombination
with pXL3749 were screened by PCR and subsequent restriction with
HindIII and sequencing to monitor the cop21 and pir116 mutations.
One clone out of the six double recombinants tested had undergone
the gene replacement. The resulting strain was named TEX1cop21.
[0263] 2) Evaluation of TEX1 cop21
[0264] TEX1cop21 was transformed by various pCOR plasmids,
including pXL2979, a 2.5 kb KmR pCOR (See Example 7.1), and was
assayed for increased copy number by gel electrophoresis. Such an
experiment with pXL2979 is shown in FIG. 17. Plasmid DNA from four
independent clones for each strain prepared with Promega miniprep
kit was linearized with EcoRI restriction enzyme, electrophoresed
on agarose gel and then stained with ethidium bromide. Each sample
represented a similar amount of bacteria, as measured by optical
density at 600 nm. The agarose gel electrophoresis obtained for the
pCOR plasmid pXL2979 produced in E. coli TEX1cop21, XAC1pir, and
TEX1 is displayed in FIG. 17. It clearly shows there was no
increase in plasmid copy number when the plasmids are produced in
the TEX1 cop21 strain, as compared with TEX1.
Example 12
Construction of TEX2pir42 (XAC-1 pir116 pir42 recA.sup.-)
[0265] Firstly, a recA-derivative of TEX1 was constructed. The
pir42 mutation was then introduced into the resulting strain named
TEX2 to generate TEX2pir42.
[0266] 1) Construction of E. coli TEX2, a recA-Derivative of
TEX1
[0267] A deleted recA gene containing 3 translation stop codons
(one in each frame) at its 5' end was obtained by PCR. This deleted
recA gene was introduced by gene replacement (Blum et al., J.
Bacteriol., 1989, 171, pp. 538-46) into the TEX1 genome. The
construction of the suicide vector for homologous recombination is
shown in FIG. 18.
[0268] PCR primers used for the amplification of recA fragments
were the following Table 6:
TABLE-US-00019 TABLE 6 primers DNA sequences seq 10930
5'CCCTCTAGATCGATAGCCATTTTTACTCCTG 3' SEQ ID seq 10931
5'CGGGATCCTGATTATGCCGTGTCTATTAG 3' SEQ ID seq 10932
5'CCCAAGCTTCTTCGTTAGTTTCTGCTACGCCTTCGC SEQ ID 3' seq 10933
5'GGTCTAGAACGTGAAAGTGGTGAAGAACAAAATCG 3' SEQ ID
[0269] Restriction sites added to the recA sequence are
underlined.
[0270] In order to maintain the RecA+ phenotype necessary for
homologous recombination to occur, the recA function was provided
to E. coli TEX1 with a plasmid containing a heterologous recA gene
that can complement E. coli recA mutants, such as for example the
recA gene of the bacterium Agrobacterium radiobacter, and an
antibiotic gene resistance, such as the ampicillin resistance gene.
After gene replacement, the plasmid was eliminated from the
recombinant strain by culture-dilution in non-selective medium
(LB). The absence of the plasmid was screened for the loss of
antibiotic resistance.
[0271] The resulting strain was named TEX2. Gene replacement was
monitored by PCR in FIG. 19. PCR Primers are described in the
following Table 7.
TABLE-US-00020 TABLE 7 Primers 11355-11354 Primers 11355-11354 Wild
type recA 1117 bp 1089 bp Deleted recA 404 bp 376 bp
[0272] The first primer was based on the sequence of the recA gene.
The second one was based on a sequence close to but outside the
homology region present in the suicide vector pXL3457 (immediately
5' or 3' of recA) to ensure that amplification can only occur on a
genomic fragment. The sequence of both oligonucleotides was chosen
according to the sequence of E. coli which comprises the recA locus
(Genbank ECAE000354).
[0273] The PCR fragments obtained from a recA-deleted strain are
shorter as compared to those obtained with a wild-type strain, as
presented in the following Table 8.
TABLE-US-00021 TABLE 8 PCR primers for amplification of recA
primers 5' -> 3' sequence seq 11352- GCGACCCTTGTGTATCAAAC SEQ ID
NO: 17 seq 11353- GGTATTACCCGGCATGACAG SEQ ID NO: 18 seq 11355-
GTGGTGGAAATGGCGATAGG SEQ ID NO: 19 seq 11354- GCGATTTTGTTCTTCACCAC
SEQ ID NO: 20
[0274] The PCR profile obtained was as expected and demonstrated
the presence of a truncated recA gene in the genome of TEX2. RecA-
phenotype (sensitivity to UV light), as well as phenotypic
characteristics of TEX2 were checked. Phenotypic characteristics of
TEX2 were the same as those of TEX1 strain, i.e., ara-, Rif.sup.R,
Nal.sup.R, Sp.sup.R, UidA-, Arg-, Km.sup.S Amp.sup.S), as
expected.
[0275] B) Construction of E. coli TEX2pir42
[0276] A TEX2pir42 strain was constructed by double homologous
recombination, according to the strategy described in Example 10,
with the exception that recombination in TEX2 was carried out in
presence of a plasmid carrying a heterologous recA gene capable of
complementing the E. coli recA mutants, in order to maintain a
RecA+ phenotype required for homologous recombination.
[0277] Gene replacement was monitored by restriction analysis of
the PCR product digested with ClaI (see FIG. 14). Gene replacement
had occurred in two out of the four-studied double recombinant
clones.
C) Evaluation of E. coli TEX2pir42
[0278] 1) Evaluation at Lab Scale Plasmid Production:
[0279] TEX2pir42 was transformed by the pCOR plasmid pXL3179 (2.4
kb). Production of pXL3179 in TEX2 was intensively studied at the
lab scale, in terms of reproducibility of the improvement of
plasmid copy number, conditions of culture, as well as stability
(number of generations). All the studies consistently showed a 2 to
5-fold increase of plasmid copy number as compared to production of
pXL3179 in TEX1 in the same conditions. Plasmid copy number was
assessed further to the production of pXL3179 in TEX2pir2, and TEX
pir42 and TEX1 as comparative experiments. In this experiment,
plasmids were extracted from identical bacterial biomass, based on
the OD at 600 nm, and analyzed by agarose gel electrophoresis. The
gel was stained with ethidium bromide after electrophoresis. The
agarose gel electrophoresis, which is displayed in FIG. 20, clearly
shows that plasmids are produced in TEX2pir42 at high copy number,
and advantageously shows that plasmid multimers are reduced when
produced in TEX2pir42 instead of TEX1pir42.
[0280] 2) Evaluation in Fermentors:
[0281] These results were confirmed at a larger scale in 7-liter
fermentors, as described below.
[0282] a) Composition of Fermentation Media
[0283] The composition of the medium used for inoculum cultures
was: Na.sub.2HPO.sub.4 6 g/l, KH.sub.2PO.sub.4 3 g/l, NaCl 0.5 g/l,
NH.sub.4Cl 1 g/l, NH.sub.4H.sub.2PO.sub.4 3 g/l, glucose 5 g/l,
MgSO.sub.4, 7H.sub.20 0.24 g/l, CaCl.sub.2,2H.sub.2O 0.015 g/l,
thiamine HCl 0.010 g/l.
[0284] The composition of the medium used for fed-batch culture was
as follows: KH.sub.2PO.sub.4 8 g/l, K.sub.2HPO.sub.4 6.3 g/l,
Na.sub.2HPO.sub.4 1.7 g/l, NH.sub.4Cl 2.5 g/l, glucose 10 g/l,
MgSO.sub.4, 7H.sub.20 2.6 g/l, thiamine 0.011 g/l, Biospumex36
antifoam 0.1 ml/l, salt mix (see table 9) 2.5 ml/l.
TABLE-US-00022 TABLE 9 Composition of salt mix Final Salt mix
concentration Solution in fed-batch (g/100 ml) medium
FeSO.sub.4,7H.sub.2O 1.6 40 CaCl.sub.2,2H.sub.2O 1.6 40
MnSO.sub.4,H.sub.2O 0.4 10 CoCl.sub.2,H.sub.2O 0.16 4
ZnSO.sub.4,7H.sub.2O 0.08 2 MoO.sub.4Na.sub.2,2H.sub.2O 0.072 1.8
CuCl.sub.2,2H.sub.2O 0.04 1 H.sub.3BO.sub.3 0.02 0.5
AlCl.sub.3,6H.sub.2O 0.04 1
The composition of the supply medium was as follows: 50% glucose,
0.7%, magnesium 0.02% thiamine-HCl, 1% Biospumex36 antifoam.
[0285] b) Fermentation Parameters
[0286] A 7-liter fermentor containing 3 liters of the fed-batch
medium was inoculated with 1.2% of the inoculum culture. Inoculum
was prepared as follows: 250 ml of the inoculum medium in a 2-liter
flask was inoculated with 0.25 ml of a frozen cell suspension of
the E. coli strain TEX2pir42 (pXL3179).
[0287] Flasks were incubated for 24 hours at 37.degree. C. at 220
rpm. After 24 hours, different parameters were measured: residual
glucose: 0 g/l, OD.sub.600 nm was 2.7 and pH 6.24. During
fermentation, the pH was controlled and adjusted automatically
between 6.9 and 7 with NH.sub.3. The temperature was maintained at
37.degree. C. and the dissolved oxygen adjusted to a 45% pO2 by
retroaction on the stirring rate.
[0288] After initial batch culturing of the bacterial strain for
about 17 hours and consumption of the carbon source (glucose), the
supply medium was added. Glucose and acids such lactate and acetate
were maintained at a concentration close to 0.
[0289] c) Results
[0290] Final results are presented in Table 10, as compared to
production in a 100-liter fermentor with E. coli TEX1 (pXL3179) in
optimized conditions. As for XAC-1pir116, there was no difference
between 7-liter and 800-liter fermentors in terms of plasmid copy
number of pXL3179 produced in TEX 10.
[0291] Plasmids pXL3179 so produced in a 7-liter fermentor using a
E. coli TEX2pir42 was compared to the production of pXL3179 in a
100-liter fermentor with E. coli TEX1, in optimized conditions. It
was demonstrated that as for the XAC-1 pir116 (See Example 5.3),
there is a stable plasmid production rate in a 7-liter, 100-liter,
or 800-liter fermentor in TEX1.
TABLE-US-00023 TABLE 10 Characteristics of the fermentation of TEX1
and TEX2pir42 strains containing pXL3179 Estimated Duration
Concentration copy of Final Cell of number fermentation OD Dry DNA
(copy/ Reference (h) (600 nm) weight (mg/l) bacterium) TEX1
OpGen090 43.00 104 33.1 96 616-627 (pXL3179) TEX2pir42 Op1328S5
48.47 72 27.1 205 1896-1904 (pXL3179)
[0292] There were 3-fold more copies of plasmid pXL3179 per
bacterium in TEX2pir42 as compared to TEX1.
[0293] Plasmids corresponding to different fermentation time points
were extracted from identical bacterial biomass, based on the OD at
600 nm, and analyzed by agarose gel electrophoresis (FIG. 21). FIG.
21 clearly shows an increase of the plasmid copy number with the
duration of the fermentation. Also, FIG. 21 shows the topology of
the pXL3179 plasmid produced in Op1328S5 TEX2pir42, which was
nearly exclusively in a monomeric form.
[0294] In conclusion, the E. coli host strain TEX2pir42 according
to the present invention provided an unexpectedly high plasmid copy
number improvement of pCOR plasmids, such as pXL3179, of 2 to
5-fold in TEX2pir42 as compared to TEX1, at a lab scale and in
fermentors. Furthermore, while the plasmid copy number was greatly
improved, plasmids so produced exhibited a monomeric topology, not
only at lab-scale but also at a larger scale (7-liter fermentor)
compatible with industrial production.
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Sequence CWU 1
1
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