Circular DNA expression cassettes for in vivo gene transfer

Double-stranded DNA molecules characterised in that they are circular and in that they essentially include one or more genes of interest.

Gene therapy consists in correcting a deficiency or an abnormality by 
introducing genetic information into the affected cell or organ. 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 
tissue concerned. Being a high molecular weight, negatively charged 
molecule, DNA has difficulties in passing spontaneously through the 
phospholipid cell membranes. Different vectors are hence used in order to 
permit gene transfer: viral vectors on the one hand, natural or synthetic, 
chemical and/or biochemical vectors on the other hand. Viral vectors 
(retroviruses, adenoviruses, adeno-associated viruses, etc.) are very 
effective, in particular in passing through membranes, but present a 
number of risks, such as pathogenicity, recombination, replication, 
immunogenicity, etc. Chemical and/or biochemical vectors enable these 
risks to be avoided (for reviews, see Behr, 1993, Cotten and Wagner, 
1993). These vectors are, for example, cations (calcium-phosphate, 
DEAE-dextran, etc.) which act by forming precipitates with DNA, which 
precipitates can be "phagocytosed" by the cells. They can also be 
liposomes in which DNA is incorporated and which fuse with the plasma 
membrane. Synthetic gene transfer vectors are generally lipids or cationic 
polymers which complex DNA and form a particle therewith carrying positive 
surface charges. These particles are capable of interacting with the 
negative charges of the cell membrane and then of crossing the latter. 
Dioctadecylamidoglycylspermine (DOGS, Transfectam.TM.) or 
N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA, 
Lipofectin.TM.) may be mentioned as examples of such vectors. Chimeric 
proteins have also been developed: they consist of a polycationic portion 
which condenses DNA, linked to a ligand which binds to a membrane receptor 
and carries the complex into the cells by endocytosis. It is thus 
theoretically possible to "target" a tissue or certain cell populations so 
as to improve the in vivo bioavailability of the transferred gene. 
However, the use of chemical and/or biochemical vectors or of naked DNA 
implies the possibility of producing large amounts of DNA of 
pharmacological purity. In effect, in these gene therapy techniques, the 
medicinal product consists of the DNA itself, and it is essential to be 
able to manufacture, in appropriate amounts, DNAs having suitable 
properties for therapeutic use in man. 
The plasmids currently 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 required for their expression (enhancer(s), promoter(s), 
polyadenylation sequences, etc.). These plasmids currently used in gene 
therapy (in clinical trials such as the treatment of melanomas, Nabel et 
al., 1992, or in experimental studies) display, however, some drawbacks 
associated, in particular, with their dissemination in the body. Thus, as 
a result of this dissemination, a competent bacterium present in the body 
can, at a low frequency, receive this plasmid. The chance of this 
occurring is all the greater for the fact that the treatment in question 
entails in vivo gene therapy in which the DNA may be disseminated in the 
patient's body and may come into contact with bacteria which infect this 
patient or alternatively with bacteria of the commensal flora. If the 
bacterium which is a recipient of the plasmid is an enterobacterium such 
as E. coli, this plasmid may replicate. Such an event then leads to the 
dissemination of the therapeutic gene. Inasmuch 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 lacking in 
the host and hence enables a genetic defect to be corrected, the 
dissemination of some of these genes could have unforeseeable and worrying 
effects (for example if a pathogenic bacterium were to acquire the gene 
for a human growth factor). Furthermore, the plasmids used in non-viral 
gene therapy also possess a marker for resistance to an antibiotic 
(ampicillin, kanamycin, etc.). Hence the bacterium acquiring such a 
plasmid has an undeniable selective advantage, since any therapeutic 
antibiotic treatment using an antibiotic of the same family as the one 
selecting the resistance gene of the plasmid will lead to the selection of 
the plasmid in question. In this connection, ampicillin belongs to the 
.beta.-lactams, which is the family of antibiotics most widely used in the 
world. It is hence necessary to seek to limit as far as possible the 
dissemination of the therapeutic genes and the resistance genes. Moreover, 
the genes carried by the plasmid, corresponding to the vector portion of 
the plasmid (function(s) required for replication, resistance gene), also 
run the risk of being expressed in the transfected cells. There is, in 
effect, a transcription background, which cannot be ruled out, due to the 
host's expression signals on the plasmid. This expression of exogenous 
proteins may be thoroughly detrimental in a number of gene therapy 
treatments, as a result of their potential immunogenicity and hence of the 
attack of the transfected cells by the immune system. 
Hence it is especially important to be able to have at one's disposal 
medicinal DNA molecules having a genetic purity suitable for therapeutic 
use. It is also especially important to have at one's disposal methods 
enabling these DNA molecules to be prepared in amounts appropriate for 
pharmaceutical use. The present invention provides a solution to these 
problems. 
The present invention describes, in effect, DNA molecules which can be used 
in gene therapy, having greatly improved genetic purity and impressive 
properties of bioavailability. The invention also describes an especially 
effective method for the preparation of these molecules and for their 
purification. 
The present invention lies, in particular, in the development of DNA 
molecules which can be used in gene therapy, virtually lacking any 
non-therapeutic region. The DNA molecules according to the invention, also 
designated minicircles on account of their circular structure, their small 
size and their supercoiled form, display many advantages. 
They make it possible, in the first place, to eliminate the risks 
associated with dissemination of the plasmid, such as (1) replication and 
dissemination which may lead to an uncontrolled overexpression of the 
therapeutic gene, (2) the dissemination and expression of resistance 
genes, and (3) the expression of genes present in the non-therapeutic 
portion of the plasmid, which are potentially immunogenic and/or 
inflammatory, and the like. The genetic information contained in the DNA 
molecules according to the invention is limited, in effect, essentially to 
the therapeutic gene(s) and to the signals for regulation of its/their 
expression (neither origin of replication, nor gene for resistance to an 
antibiotic, and the like). The probability of these molecules (and hence 
of the genetic information they contain) being transferred to a 
microorganism and being stably maintained is almost zero. 
Furthermore, due to their small size, DNA molecules according to the 
invention potentially have better bioavailability in vivo. In particular, 
they display improved capacities for cell penetration and cellular 
distribution. Thus, it is recognized that the coefficient of diffusion in 
the tissues is inversely proportional to the molecular weight (Jain, 
1987). Similarly, at cellular level, high molecular weight molecules have 
inferior permeability through the plasma membrane. In addition, for the 
plasmid to progress to the nucleus, which is essential for its expression, 
high molecular weight is also a drawback, the nuclear pores imposing a 
size limit for diffusion to the nucleus (Landford et al., 1986). The 
elimination of the non-therapeutic portions of the plasmid (origin of 
replication and resistance gene in particular) according to the invention 
also enables the size of the DNA molecules to be decreased. This decrease 
may be estimated at a factor of 2, reckoning, for example, 3 kb for the 
origin of replication and the resistance marker (vector portion) and 3 kb 
for the transgene with the sequences required for its expression. This 
decrease (i) in molecular weight and (ii) in negative charge endows the 
molecules of the invention with improved capacities for tissue, cellular 
and nuclear diffusion and bioavailability. 
Hence a first subject of the invention lies in a double-stranded DNA 
molecule having the following features: it is circular in shape and 
essentially comprises one or more genes of interest. As stated above, the 
molecules of the invention essentially lack non-therapeutic regions, and 
especially an origin of replication and/or a marker gene. In addition, 
they are advantageously in supercoiled form. 
The present invention is also the outcome for the development of a method, 
of constructions and of cell hosts which are specific and especially 
effective for the production of these therapeutic DNA molecules. More 
especially, the method according to the invention lies in the production 
of therapeutic DNA molecules defined above, by excision from a plasmid or 
from a chromosome by site-specific recombination. The method according to 
the invention is especially advantageous, since it does not necessitate a 
prior step of purification of the plasmid, is very specific, especially 
effective, does not decrease the amounts of DNA produced and leads 
directly to therapeutic molecules of very great genetic purity and of 
great bioavailability. This method leads, in effect, to the generation of 
circular DNA molecules (minicircles) essentially containing the gene of 
interest and the regulator sequences permitting its expression in the 
cells, tissue, organ or apparatus, or even the whole body, in which the 
expression is desired. In addition, these molecules may then be purified 
by standard techniques. 
The site-specific recombination may be carried out by means of various 
systems which lead to site-specific recombination between sequences. More 
preferably, the site-specific recombination in the method of the invention 
is obtained by means of two specific sequences which are capable of 
recombining with one another in the presence of a specific protein, 
generally designated recombinase. For this reason, the DNA molecules 
according to the invention generally comprise, in addition, a sequence 
resulting from this site-specific recombination. The sequences permitting 
the recombination used in the context of the invention generally comprise 
from 5 to 100 base pairs, and more preferably fewer than 50 base pairs. 
The site-specific recombination may be carried out in vivo (that is to say 
in the host cell) or in vitro (that is to say on a plasmid preparation). 
In this connection, the present invention also provides particular genetic 
constructions suitable for the production of the therapeutic DNA molecules 
defined above. These genetic constructions, or recombinant DNAs, according 
to the invention comprise, in particular, the gene or genes of interest 
flanked by the two sequences permitting site-specific recombination, 
positioned in the direct orientation. The position in the direct 
orientation indicates that the two sequences follow the same 5'-3' 
polarity in the recombinant DNA according to the invention. The genetic 
constructions of the invention can be double-stranded DNA fragments 
(cassettes) essentially composed of the elements mentioned above. These 
cassettes can be used for the construction of cell hosts having these 
elements integrated in their genome (FIG. 1). The genetic constructions of 
the invention can also be plasmids, that is to say any linear or circular 
DNA molecule capable of replicating in a given host cell, containing the 
gene or genes of interest flanked by the two sequences permitting 
site-specific recombination, positioned in the direct orientation. The 
construction can be, more specifically, a vector (such as a cloning and/or 
expression vector), a phage, a virus, and the like. These plasmids of the 
invention may be used to transform any competent cell host for the purpose 
of the production of minicircles by replication of the plasmid followed by 
excision of the minicircle (FIG. 2). 
In this connection, another subject of the invention lies in a recombinant 
DNA comprising one or more genes of interest, flanked by two sequences 
permitting site-specific recombination, positioned in the direct 
orientation. 
The recombinant DNA according to the invention is preferably a plasmid 
comprising at least: 
a) an origin of replication and optionally a marker gene, 
b) two sequences permitting a site-specific recombination, positioned in 
the direct orientation, and, 
c) placed between said sequences b), one or more genes of interest. 
The specific recombination system present in the genetic constructions 
according to the invention can be of different origins. In particular, the 
specific sequences and the recombinases used can belong to different 
structural classes, and in particular to the integrase family of 
bacteriophage .lambda. or to the resolvase family of the transposon Tn3. 
Among recombinases belonging to the integrase family of bacteriophage 
.lambda., there may be mentioned, in particular, the integrase of the 
phages lambda (Landy et al., Science 197 (1977) 1147), P22 and .PHI.80 
(Leong et al., J. Biol. Chem. 260 (1985) 4468), HP1 of Haemophilus 
influenza (Hauser et al., J. Biol. Chem. 267 (1992) 6859), the Cre 
integrase of phage P1, the integrase of the plasmid pSAM2 (EP 350,341) or 
alternatively the FLP recombinase of the 2.mu. plasmid. When the DNA 
molecules according to the invention are prepared by recombination by 
means of a site-specific system of the integrase family of bacteriophage 
lambda, the DNA molecules according to the invention generally comprise, 
in addition, a sequence resulting from the recombination between two att 
attachment sequences of the corresponding bacteriophage or plasmid. 
Among recombinases belonging to the family of the transposon Tn3, there may 
be mentioned, in particular, the resolvase of the transposon Tn3 or of the 
transposons Tn21 and Tn522 (Stark et al., 1992); the Gin invertase of 
bacteriophage mu or alternatively the resolvase of plasmids, such as that 
of the par fragment of RP4 (Albert et al., Mol. Microbiol. 12 (1994) 131). 
When the DNA molecules according to the invention are prepared by 
recombination by means of a site-specific system of the family of the 
transposon Tn3, the DNA molecules according to the invention generally 
comprise, in addition, a sequence resulting from the recombination between 
two recognition sequences of the resolvase of the transposon in question. 
According to a particular embodiment, in the genetic constructions of the 
present invention, the sequences permitting site-specific recombination 
are derived from a bacteriophage. More preferably, these latter are 
attachment sequences (attP and attB sequences) of a bacteriophage, or 
derived sequences. These sequences are capable of recombining specifically 
with one another in the presence of a recombinase designated integrase. 
The term derived sequence includes the sequences obtained by 
modification(s) of the attachment sequences of the bacteriophages, which 
retain the capacity to recombine specifically in the presence of the 
appropriate recombinase. Thus, such sequences can be reduced fragments of 
these sequences or, on the contrary, fragments extended by the addition of 
other sequences (restriction sites, and the like). They can also be 
variants obtained by mutation(s), in particular by point mutation(s). The 
terms attP and attB sequences of a bacteriophage or of a plasmid denote, 
according to the invention, the sequences of the recombination system 
specific to said bacteriophage or plasmid, that is to say the attP 
sequence present in said phage or plasmid and the corresponding 
chromosomal attB sequence. 
By way of preferred examples, there may be mentioned, in particular, the 
attachment sequences of the phages lambda, P22, .PHI.80, P1 and HP1 of 
Haemophilus influenzae or alternatively of plasmid pSAM2 or the 2.mu. 
plasmid. These sequences are advantageously chosen from all or part of the 
sequences SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 6, SEQ ID No. 7, SEQ ID 
No. 8, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 12, SEQ ID 
No. 13 and SEQ ID No. 14. These sequences comprise, in particular, the 
central region homologous to the attachment sequences of these phages. 
In this connection, a preferred plasmid according to the present invention 
comprises 
(a) a bacterial origin of replication and optionally a marker gene, 
(b) the attP and attB sequences of a bacteriophage selected from the phages 
lambda, P22, .PHI.80, HP1 and P1 or of plasmid pSAM2 or the 2.mu. plasmid, 
or derived sequences; and, 
(c) placed between said sequences b), one or more genes of interest. 
According to an especially preferred embodiment, the sequences in question 
are the attachment sequences (attP and attB) of phage lambda. Plasmids 
carrying these sequences are, in particular, the plasmids pXL2648, pXL2649 
or pXL2650. When these plasmids are brought, in vivo or in vitro, into 
contact with the integrase of phage lambda, the sequences recombine with 
one another to generate in vivo or in vitro, by excision, a minicircle 
according to the invention essentially comprising the elements (c), that 
is to say the therapeutic portion (FIG. 2). 
Still according to a particular embodiment of the invention, the sequences 
permitting site-specific recombination are derived from the loxP region of 
phage P1. This region is composed essentially of two inverted repeat 
sequences capable of recombining specifically with one another in the 
presence of a protein, designated Cre (Sternberg et al., J. Mol. Biol. 150 
(1971) 467). In a particular variant, the invention hence relates to a 
plasmid comprising (a) a bacterial origin of replication and optionally a 
marker gene; (b) the inverted repeat sequences of bacteriophage P1 (loxP 
region); and (c), placed between said sequences (b), one or more genes of 
interest. 
According to another particular embodiment, in the genetic constructions of 
the present invention, the sequences permitting site-specific 
recombination are derived from a transposon. More preferably, the 
sequences in question are recognition sequences of the resolvase of a 
transposon, or derived sequences. By way of preferred examples, there may 
be mentioned, in particular, the recognition sequences of the transposons 
Tn3, Tn21 and Tn522. By way of a preferred example, there may be mentioned 
the sequence SEQ ID No. 15 or a derivative of the latter (see also 
Sherrat, P. 163-184, Mobile DNA, Ed. D. Berg and M. Howe, American Society 
for Microbiology, Washington D.C. 1989). 
According to another especially advantageous variant, the plasmids of the 
invention comprise, in addition, a multimer resolution sequence. This is 
preferably the mrs (multimer resolution system) sequence of the plasmid 
RK2. More preferably, the invention relates to a plasmid comprising: 
(a) a bacterial origin of replication and optionally a marker gene, 
(b) the attP and attB sequences of a bacteriophage, in the direct 
orientation, selected from the phages lambda, P22, .PHI.80, HP1 and P1 or 
of plasmid pSAM2 or the 2.mu. plasmid, or derived sequences; and, 
(c) placed between said sequences b), one or more genes of interest and the 
mrs sequence of plasmid RK2. 
This embodiment is especially advantageous. Thus, when plasmids pXL2649 or 
pXL2650 are brought into contact with the integrase of the bacteriophage 
in vivo, the sequences recombine to generate the minicircle and the 
miniplasmid, but also multimeric or topological forms of minicircle or of 
miniplasmid. It is especially advantageous to be able to decrease the 
concentration of these forms in order to increase the production and 
facilitate the purification of minicircle. 
The multimeric forms of plasmids are known to a person skilled in the art. 
For example, the cer fragment of ColE1 (Summers et al., 1984 Cell 36 p. 
1097) or the mrs site of the par locus of RK2 (L. Ebert 1994 Mol. 
Microbiol. 2 p. 131) permit the resolution of multimers of plasmids and 
participate in an enhanced stability of the plasmid. However, whereas 
resolution at the cer site requires four proteins encoded by the E. coli 
genome (Colloms et al., 1990 J. Bacteriol. 172 p. 6973), resolution at the 
mrs site requires only the ParA protein for which the parA gene is mapped 
on the par locus of RK2. As a result, it would appear advantageous to use 
all or a portion of the par locus containing parA and the mrs sequence. 
For example, the mrs sequence may be placed between the attB and attP 
sequences of phage lambda, and the parA gene be expressed in trans or in 
cis from its own promoter or from an inducible promoter. In this 
connection, a particular plasmid of the invention comprises: 
(a) a bacterial origin of replication and optionally a marker gene, 
(b) the attP and attB sequences of a bacteriophage, in the direct 
orientation, selected from the phages lambda, P22, .PHI.80, HP1 and P1 or 
of plasmid pSAM2 or the 2.mu. plasmid, or derived sequences, 
(c) placed between said sequences b), one or more genes of interest and the 
mrs sequence of plasmid RK2, and 
(d) the parA gene of plasmid RK2. 
One such plasmid is, in particular, the plasmid pXL2960 described in the 
examples. It may be employed, and can enable minicircle to be produced 
exclusively in monomeric form. 
According to another advantageous variant, the plasmids of the invention 
comprise two sets of site-specific recombination sequences from a 
different family. These advantageously comprise a first set of 
integrase-dependent sequences and a second set of parA-dependent 
sequences. The use of two sets of sequences enables the production yields 
of minicircles to be increased when the first site-specific recombination 
is incomplete. Thus, when plasmids pXL2650 or pXL2960 are brought into 
contact with the integrase of the bacteriophage in vivo, the sequences 
recombine to generate the miniplasmid and the minicircle, but this 
reaction is not complete (5 to 10% of initial plasmid may be left). The 
introduction, in proximity to each of the att sequences of phage lambda, 
of an mrs sequence of RK2 enables the production of minicircles to be 
increased. Thus, after induction of the integrase of phage lambda and 
Int-dependent recombination, the unrecombined molecules will be able to 
come under the control of the ParA protein of RK2 and to recombine at the 
mrs sites. Conversely, after induction of the ParA protein and 
ParA-dependent recombination, the unrecombined molecules will be able to 
come under the control of the integrase of phage lambda and will be able 
to recombine at the att sites. Such constructions thus make it possible to 
produce minicircle and negligible amounts of unrecombined molecules. The 
att sequences, like the mrs sequences, are in the direct orientation, and 
the int and parA genes may be induced simultaneously or successively from 
the same inducible promoter or from two inducible promoters. Preferably, 
the sequences in question are the attB and attP attachment sequences of 
phage lambda in the direct orientation and two mrs sequences of RK2 in the 
direct orientation. 
As stated above, another aspect of the present invention lies in a method 
for the production of therapeutic DNA molecules defined above, by 
excision, from a plasmid or chromosome, by site-specific recombination. 
Another subject of the present invention hence lies in a method for the 
production of a DNA molecule (minicircle) as defined above, according to 
which a culture of host cells containing a recombinant DNA as defined 
above is brought into contact with the recombinase enabling site-specific 
recombination to be induced. More preferably, the culture and recombinase 
are brought into contact either by transfection or infection with a 
plasmid or a phage containing the gene for said recombinase; or by 
induction of the expression of a gene coding for said recombinase, present 
in the host cell. As mentioned below, this gene may be present in the host 
cell in integrated form in the genome, on a replicative plasmid or 
alternatively on the plasmid of the invention, in the non-therapeutic 
portion. 
To permit the production of the minicircles according to the invention by 
site-specific recombination in vivo, the recombinase used must be 
introduced into, or induced in, cells or the culture medium at a 
particular instant. For this purpose, different methods may be used. 
According to a first method, a host cell is used containing the 
recombinase gene in a form permitting its regulated expression. It may, in 
particular, be introduced under the control of a promoter or of a system 
of inducible promoters, or alternatively in a temperature-sensitive 
system. In particular, the gene may be present in a temperature-sensitive 
phage, latent during the growth phase, and induced at a suitable 
temperature (for example lysogenic phage lambda Xis.sup.- cI857). The 
cassette for expression of the recombinase gene may be carried by a 
plasmid, a phage or even by the plasmid of the invention, in the 
non-therapeutic region. It may be integrated in the genome of the host 
cell or maintained in replicative form. According to another method, the 
cassette for expression of the gene is carried by a plasmid or a phage 
used to transfect or infect the cell culture after the growth phase. In 
this case, it is not necessary for the gene to be in a form permitting its 
regulated expression. In particular, any constitutive promoter may be 
used. The cell may also be brought into contact with the recombinase in 
vitro, on a plasmid preparation, by direct incubation with the protein. 
It is preferable, in the context of the present invention, to use a host 
cell capable of expressing the recombinase gene in a regulated manner. 
This embodiment, in which the recombinase is supplied directly by the host 
cell after induction, is especially advantageous. In effect, it suffices 
simply to place the cells in culture at the desired time under the 
conditions for expression of the recombinase gene (permissive temperature 
for a temperature-sensitive gene, addition of an inducer for a regulable 
promoter, and the like) in order to induce the site-specific recombination 
in vivo and thus the excision of the minicircle of the invention. In 
addition, this excision takes place in especially high yields, since all 
the cells in culture express the recombinase, which is not necessarily the 
case if a transfection or an infection has to be carried out in order to 
transfer the recombinase gene. 
According to a first embodiment, the method of the invention comprises the 
excision of the molecules of therapeutic DNA by site-specific 
recombination from a plasmid. This embodiment employs the plasmids 
described above permitting, in a first stage, replication in a chosen 
host, and then, in a second stage, the excision of the non-therapeutic 
portions of said plasmid (in particular the origin of replication and the 
resistance gene) by site-specific recombination, generating the circular 
DNA molecules of the invention. To carry out the method, different types 
of plasmid may be used, and especially a vector, a phage or a virus. A 
replicative vector is preferably used. 
Advantageously, the method of the invention comprises a prior step of 
transformation of host cells with a plasmid as defined above, followed by 
culturing of the transformed cells, enabling suitable amounts of plasmid 
to be obtained. Excision by site-specific recombinations is then carried 
out by bringing into contact with the recombinase under the conditions 
defined above (FIG. 2). As stated above, in this embodiment, the 
site-specific recombination may be carried out in vivo. (that is to say in 
the host cell) or in vitro (that is to say on a plasmid preparation). 
According to a preferred embodiment, the DNA molecules of the invention are 
hence obtained from a replicative vector, by excision of the 
non-therapeutic portion carrying, in particular, the origin of replication 
and the marker gene, by site-specific recombination. 
According to another embodiment, the method of the invention comprises the 
excision of the DNA molecules from the genome of the host cell by 
site-specific recombination. This embodiment is based more especially on 
the construction of cell hosts comprising, inserted into their genome, one 
or more copies of a cassette comprising the gene of interest flanked by 
the sequences permitting recombination (FIG. 1). Different techniques may 
be used for insertion of the cassette of the invention into the genome of 
the host cell. In particular, insertion at several distinct points of the 
genome may be obtained by using integrative vectors. In this connection, 
different transposition systems such as, in particular, the miniMu system 
or defective transposons such as Tn10 derivatives, for example, may be 
used (Kleckner et al., Methods Enzymol. 204 (1991) 139; Groisman E., 
Methods Enzymol. 204 (1991) 180). The insertion may also be carried out by 
homologous recombination, enabling a cassette containing two recombination 
sequences in the direct orientation flanking one or more genes of interest 
to be integrated in the genome of the bacterium. This process may, in 
addition, be reproduced as many times as desired so as to have the largest 
possible number of copies per cell. Another technique also consists in 
using an in vivo amplification system using recombination, as described in 
Labarre et al. (Labarre J., O. Reyes, Guyonvarch, and G. Leblon. 1993. 
Gene replacement, integration, and amplification at the gdhA locus of 
Corynebacterium glutamicum. J. Bacteriol. 175:1001-107), so as to augment 
from one copy of the cassette to a much larger number. 
A preferred technique consists in the use of miniMu. To this end, miniMu 
derivatives are constructed comprising a resistance marker, the functions 
required in cis for their transposition and a cassette containing two 
recombination sequences in the direct orientation flanking the gene or 
genes of interest. These miniMus are advantageously placed at several 
points of the genome using a resistance marker (kanamycin, for example) 
enabling several copies per genome to be selected (Groisman E. cited 
above). As described above, the host cell in question can also express 
inducibly a site-specific recombinase leading to the excision of the 
fragment flanked by the recombination sequences in the direct orientation. 
After excision, the minicircles may be purified by standard techniques. 
This embodiment of the method of the invention is especially advantageous, 
since it leads to the generation of a single type of plasmid molecule: the 
minicircle of the invention. The cells do not contain, in effect, any 
other episomal plasmid, as is the case during production from a plasmid 
(FIGS. 1 and 2). 
Another subject of the invention also lies in a modified host cell 
comprising, inserted into its genome, one or more copies of a recombinant 
DNA as defined above. 
The invention also relates to any recombinant cell containing a plasmid as 
defined above. These cells are obtained by any technique known to a person 
skilled in the art enabling a DNA to be introduced into a given cell. Such 
a technique can be, in particular, transformation, electroporation, 
conjugation, protoplast fusion or any other technique known to a person 
skilled in the art. As regards transformation, different protocols have 
been described in the prior art. In particular, cell transformation may be 
carried out by treating whole cells in the presence of lithium acetate and 
polyethylene glycol according to the technique described by Ito et al. (J. 
Bacteriol. 153 (1983) 163-168), or in the presence of ethylene glycol and 
dimethyl sulphoxide according to the technique of Durrens et al. (Curr. 
Genet. 18 (1990) 7). An alternative protocol has also been described in 
Patent Application EP 361,991. As regards electroporation, this may be 
carried out according to Becker and Guarentte (in: Methods in Enzymology 
Vol194 (1991) 182). 
The method according to the invention may be carried out in any type of 
cell host. Such hosts can be, in particular, bacteria or eukaryotic cells 
(yeasts, animal cells, plant cells), and the like. Among bacteria, E.coli, 
B. subtilis, Streptomyces, Pseudomonas (P. putida, P. aeruginosa), 
Rhizobium meliloti, Agrobacterium tumefaciens, Staphylococcus aureus, 
Streptomyces pristinaespiralis, Enterococcus faecium or Clostridium, and 
the like, may be mentioned more preferentially. Among bacteria, it is 
preferable to use E.coli. Among yeasts, Kluyveromyces, Saccharomyces, 
Pichia, Hansenula, and the like, may be mentioned. Among mammalian animal 
cells, CHO, COS, NIH3T3, and the like, cells may be mentioned. 
In accordance with the host used, the plasmid according to the invention is 
adapted by a person skilled in the art to permit its replication. In 
particular, the origin of replication and the marker gene are chosen in 
accordance with the host cell selected. 
The marker gene may be a resistance gene, in particular for resistance to 
an antibiotic (ampicillin, kanamycin, geneticin, hygromycin, and the 
like), or any gene endowing the cell with a function which it no longer 
possesses (for example a gene which has been deleted on the chromosome or 
rendered inactive), the gene on the plasmid reestablishing this function. 
In a particular embodiment, the method of the invention comprises an 
additional step of purification of the minicircle. 
In this connection, the minicircle may be purified by standard techniques 
of plasmid DNA purification, since it is supercoiled like plasmid DNA. 
These techniques comprise, inter alia, purification on a cesium chloride 
density gradient in the presence of ethidium bromide, or alternatively the 
use of anion exchange columns (Maniatis et al., 1989). In addition, if the 
plasmid DNA corresponding to the non-therapeutic portions (origin of 
replication and selectable marker in particular) is considered to be 
present in an excessively large amount, it is also possible, after or 
before the purification, to use one or more restriction enzymes which will 
digest the plasmid and not the minicircle, enabling them to be separated 
by techniques that separate supercoiled DNA from linear DNA, such as a 
cesium chloride density gradient in the presence of ethidium bromide 
(Maniatis et al., 1989). 
In addition, the present invention also describes an improved method for 
the purification of minicircles. This method enables minicircles of very 
great purity to be obtained in large yields in a single step. This 
improved method is based on the interaction between a double-stranded 
sequence present in the minicircle and a specific ligand. The ligand can 
be of various natures, and in particular protein, chemical or nucleic acid 
in nature. It is preferably a ligand of the nucleic acid type, and in 
particular an oligonucleotide, optionally chemically modified, capable of 
forming by hybridization a triple helix with the specific sequence present 
in the DNA molecule of the invention. It was, in effect, shown that some 
oligonucleotides were capable of specifically forming triple helices with 
double-stranded DNA sequences (Helene et al., Biochim. Biophys. Acta 1049 
(1990) 99; see also FR 94/15162 incorporated in the present application by 
reference). 
In an especially advantageous variant, the DNA molecules of the invention 
hence contain, in addition, a sequence capable of interacting specifically 
with a ligand (FIG. 3). Preferably, it is a sequence capable of forming, 
by hybridization, a triple helix with a specific oligonucleotide. This 
sequence may be positioned at any site of the DNA molecule of the 
invention, provided it does not affect the functionality of the gene of 
interest. This sequence is also present in the genetic constructions of 
the invention (plasmids, cassettes), in the portion containing the gene of 
interest (see, in particular, the plasmid pXL2650). Preferably, the 
specific sequence present in the DNA molecule of the invention comprises 
between 5 and 30 base pairs. 
The oligonucleotides used for carrying out the method according to the 
invention can contain the following bases: 
thymidine (T), which is capable of forming triplets with A.T doublets of 
double-stranded DNA (Rajagopal et al., Biochem 28 (1989) 7859); 
adenine (A), which is capable of forming triplets with A.T doublets of 
double-stranded DNA; 
guanine (G), which is capable of forming triplets with G.C doublets of 
doubled-stranded DNA; 
protonated cytosine (C+), which is capable of forming triplets with G.C 
doublets of doubled-stranded DNA (Rajagopal et al., cited above). 
Preferably, the oligonucleotide used comprises a homopyrimidine sequence 
containing cytosines, and the specific sequence present in the DNA 
molecule is a homopurine-homopyrimidine sequence. The presence of 
cytosines makes it possible to have a triple helix which is stable at acid 
pH where the cytosines are protonated, and destabilized at alkaline pH 
where the cytosines are neutralized. 
To permit the formation of a triple helix by hybridization, it is important 
for the oligonucleotide and the specific sequence present in the DNA 
molecule of the invention to be complementary. In this connection, to 
obtain the best yields and best selectivity, an oligonucleotide and a 
specific sequence which are fully complementary are used in the method of 
the invention. Possible combinations are, in particular, a poly(CTT) 
oligonucleotide and a poly(GAA) specific sequence. By way of example, 
there may be mentioned the oligonucleotide of sequence 
GAGGCTTCTTCTTCTTCTTCTTCTT (SEQ ID No. 5), in which the bases GAGG do not 
form a triple helix but enable the oligonucleotide to be spaced apart from 
the coupling arm. 
It is understood, however, that some mismatches may be tolerated, provided 
they do not lead to too great a loss of affinity. The oligonucleotide used 
may be natural (composed of unmodified natural bases) or chemically 
modified. In particular, the oligonucleotide may advantageously possess 
some chemical modifications enabling its resistance or its protection 
against nucleases, or its affinity for the specific sequence, to be 
increased. 
Thus, the oligonucleotide may be rendered more resistant to nucleases by 
modification of the skeleton (e.g. methylphosphonates, phosphorothiates, 
phosphotriester, phosphoramidate, and the like). Another type of 
modification has as its objective, more especially, to improve the 
interaction and/or the affinity between the oligonucleotide and the 
specific sequence. In particular, a thoroughly advantageous modification 
according to the invention consists in methylating the cytosines of the 
oligonucleotide. The oligonucleotide thus methylated displays the 
noteworthy property of forming a stable triple helix with the specific 
sequence at neutral pH. Hence it makes it possible to work at higher pH 
values than the oligonucleotides of the prior art, that is to say at pH 
values where the risks of degradation of the plasmid DNA are lower. 
The length of the oligonucleotide used in the method of the invention is at 
least 3 bases, and preferably between 5 and 30. An oligonucleotide of 
length greater than 10 bases is advantageously used. The length may be 
adapted to each individual case by a person skilled in the art in 
accordance with the desired selectivity and stability of the interaction. 
The oligonucleotides according to the invention may be synthesized by any 
known technique. In particular, they may be prepared by means of nucleic 
acid synthesizers. It is quite obvious that any other method known to a 
person skilled in the art may be used. 
To carry out the method of the invention, the specific ligand (protein, 
nucleic acid, and the like) may be grafted or otherwise onto a support. 
Different types of supports may be used for this purpose, such as, in 
particular, functionalized chromatography supports, in bulk form or 
prepacked in columns, functionalized plastic surfaces or functionalized 
latex beads, magnetic or otherwise. Chromatography supports are preferably 
used. By way of example, the chromatography supports which may be used are 
agatose, acrylamide or dextran, as well as their derivatives (such as 
Sephadex, Sepharose, Superose, etc.), polymers such as 
poly(styrenedivinylbenzene), or grafted or ungrafted silica, for example. 
The chromatography columns can function in the diffusion or perfusion 
mode. 
To permit its covalent coupling to the support, the ligand is generally 
functionalized. In the case of an oligonucleotide, this may be modified, 
for example, with a terminal thiol, amine or carboxyl group at the 5' or 
3' position. In particular, the addition of a thiol, amine or carboxyl 
group makes it possible, for example, to couple the oligonucleotide to a 
support carrying disulphide, maleimide, amine, carboxyl, ester, epoxide, 
cyanogen bromide or aldehyde functions. These couplings form by the 
establishment of disulphide, thioether, ester, amide or amine links 
between the oligonucleotide and the support. Any other method known to a 
person skilled in the art may be used, such as bifunctional coupling 
reagents, for example. 
Moreover, to improve the activity of the coupled oligonucleotide, it may be 
advantageous to perform the coupling by means of an "arm". Use of an arm 
makes it possible, in effect, to bind the oligonucleotide at a chosen 
distance from the support, enabling its conditions of interaction with the 
DNA molecule of the invention to be improved. The arm advantageously 
consists of nucleotide bases that do not interfere with the hybridization. 
Thus, the arm may comprise purine bases. By way of example, the arm may 
comprise the sequence GAGG. 
The DNA molecules according to the invention may be used in any application 
of vaccination or of gene and cell therapy, for the transfer of a gene to 
a body, a tissue or a given cell. In particular, they may be used for a 
direct administration in vivo, or for the modification of cells in vitro 
or ex vivo with a view to their implantation in a patient. In this 
connection, the molecules according to the invention may be used as they 
are (in the form of naked DNA), or in combination with different synthetic 
or natural, chemical and/or biochemical vectors. The latter can be, in 
particular, cations (calcium phosphate, DEAE-dextran, etc.) which act by 
forming precipitates with DNA, which precipitates can be "phagocytosed" by 
the cells. They can also be liposomes in which the DNA molecule is 
incorporated and which fuse with the plasma membrane. Synthetic gene 
transfer vectors are generally lipids or cationic polymers which complex 
DNA and form a particle therewith carrying positive surface charges. These 
particles are capable of interacting with the negative charges of the cell 
membrane and then of crossing the latter. DOGS (Transfectam.TM.) or DOTMA 
(Lipofectin.TM.) may be mentioned as examples of such vectors. Chimeric 
proteins have also been developed: they consist of a polycationic portion 
which condenses DNA, linked to a ligand which binds to a membrane receptor 
and carries the complex into the cells by endocytosis. The DNA molecules 
according to the invention may also be used for gene transfer into cells 
by physical transfection techniques such as bombardment, electroporation, 
and the like. In addition, prior to their therapeutic use, the molecules 
of the invention may optionally be linearized, for example by enzymatic 
cleavage. 
In this connection, another subject of the present invention relates to any 
pharmaceutical composition comprising at least one DNA molecule as defined 
above. This molecule may be naked or combined with a chemical and/or 
biochemical transfection vector. The pharmaceutical compositions according 
to the invention may be formulated with a view to topical, oral, 
parenteral, intranasal, intravenous, intramuscular, subcutaneous, 
intra-ocular, transdermal, and the like, administration. Preferably, the 
DNA molecule is used in an injectable form or by application. It may be 
mixed with any pharmaceutically acceptable vehicle for an injectable 
formulation, in particular for a direct injection at the site to be 
treated. The compositions can be, in particular, in the form of isotonic 
sterile solutions, or of dry, in particular lyophilized compositions 
which, on addition of sterilized water or physiological saline as 
appropriate, enable injectable solutions to be made up. Diluted Tris or 
PBS buffers in glucose or sodium chloride may be used in particular. A 
direct injection of the nucleic acid into the affected region of the 
patient is advantageous, since it enables the therapeutic effect to be 
concentrated in the tissues affected. The doses of nucleic acid used may 
be adapted in accordance with different parameters, and in particular in 
accordance with the gene, the vector, the mode of administration used, the 
pathology in question or alternatively the desired treatment period. 
The DNA molecules of the invention may contain one or more genes of 
interest, that is to say one or more nucleic acids (cDNA, gDNA, synthetic 
or semi-synthetic DNA, and the like) whose transcription and, where 
appropriate, translation in the target cell generate products of 
therapeutic, vaccinal, agricultural or veterinary value. 
Among the genes of therapeutic value, there may be mentioned, more 
especially, the genes coding for enzymes, blood derivatives, hormones, 
lymphokines, namely interleukins, interferons, TNF, and the like (FR 
92/03120), growth factors, neurotransmitters or their precursors or 
synthetic enzymes, trophic factors, namely BDNF, CNTF, NGF, IGF, GMF, 
aFGF, bFGF, NT3, NT5, and the like; apolipoproteins, namely ApoAI, ApoAIV, 
ApoE, and the like (FR 93/05125), dystrophin or a minidystrophin (FR 
91/11947), tumour suppressive genes, namely p53, Rb, Rap1A, DCC, k-rev, 
and the like (FR 93/04745), genes coding for factors involved in 
coagulation, namely factors VII, VIII, IX, and the like, suicide genes, 
namely thymidine kinase, cytosine deaminase, and the like; or 
alternatively all or part of a natural or artificial immunoglobulin (Fab, 
ScFv, and the like), a ligand RNA (WO91/19813), and the like. The 
therapeutic gene can also be an antisense gene or sequence whose 
expression in the target cell enables gene expression or the transcription 
of cellular mRNAs to be controlled. Such sequences can, for example, be 
transcribed in the target cell into RNAs complementary to cellular mRNAs, 
and can thus block their translation into protein, according to the 
technique described in Patent EP 140,308. 
The gene of interest can 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 vaccine production. Such antigenic 
peptides can be, in particular, those specific to the Epstein-Barr virus, 
the HIV virus, the hepatitis B virus (EP 185,573) or the pseudorabies 
virus, or alternatively tumour-specific peptides (EP 259,212). 
Generally, in the plasmids and molecules of the invention, the gene of 
therapeutic, vaccinal, agricultural or veterinary value also contains a 
transcription promoter region which is functional in the target cell or 
body (i.e. mammals), as well as a region located at the 3' end and which 
specifies a transcription termination signal and a polyadenylation site 
(expression cassette). As regards the promoter region, this can be a 
promoter region naturally responsible for the expression of the gene in 
question when the latter is capable of functioning in the cell or body in 
question. The promoter regions can also be those of different origin 
(responsible for the expression of other proteins, or even synthetic 
promoters). In particular, the promoter sequences can be from eukaryotic 
or viral genes. For example, they can be promoter sequences originating 
from the genome of the target cell. Among eukaryotic promoters, it is 
possible to use any promoter or derived sequence that stimulates or 
represses the transcription of a gene, specifically or otherwise, 
inducibly or otherwise, strongly or weakly. They can be, in particular, 
ubiquitous promoters (promoter of the HPRT, PGK, .alpha.-actin, tubulin, 
and the like, genes), promoters of intermediate filaments (promoter of the 
GFAP, desmin, vimentin, neurofilament, keratin, and the like, genes), 
promoters of therapeutic genes (for example the promoter of the MDR, CFTR, 
factor VIII, ApoAI, and the like, genes), tissue-specific promoters 
(promoter of the pyruvate kinase gene, villin gene, gene for intestinal 
fatty acid binding protein, gene for .alpha.-actin of smooth muscle, and 
the like) or alternatively promoters that respond to a stimulus (steroid 
hormone receptor, retinoic acid receptor, and the like). Similarly, the 
promoter sequences may be those originating 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 RSV LTR promoter, and the 
like. In addition, these promoter regions may be modified by the addition 
of activator or regulator sequences or sequences permitting a 
tissue-specific or -preponderant expression. 
Moreover, the gene of interest can also contain a signal sequence directing 
the synthesized product into the pathways of secretion of the target cell. 
This signal sequence can be the natural signal sequence of the product 
synthesized, but it can also be any other functional signal sequence, or 
an artificial signal sequence. 
Depending on the gene of interest, the DNA molecules of the invention may 
be used for the treatment or prevention of a large number of pathologies, 
including genetic disorders (dystrophy, cystic fibrosis, and the like), 
neurodegenerative diseases (Alzheimer's, Parkinson's, ALS, and the like), 
cancers, pathologies associated with disorders of coagulation or with 
dyslipoproteinaemias, pathologies associated with viral infections 
(hepatitis, AIDS, and the like), or in the agricultural and veterinary 
fields, and the like. 
The present invention will be described more completely by means of the 
examples which follow, which are to be regarded as illustrative and 
non-limiting.

General techniques of cloning and molecular biology. 
The standard methods of molecular biology, such as centrifugation of 
plasmid DNA in a cesium chloride-ethidium bromide gradient, digestion with 
restriction enzymes, gel electrophoresis, electroelution of DNA fragments 
from agarose gels, transformation in E.coli, precipitation of nucleic 
acids, and the like, are described in the literature (Maniatis et al., 
1989, Ausubel et al., 1987). Nucleotide sequences were determined by the 
chain termination method according to the protocol already put forward 
(Ausubel et al., 1987). 
Restriction enzymes were supplied by New-England Biolabs (Biolabs), 
Bethesda Research Laboratories (BRL) or Amersham Ltd. (Amersham). 
To carry out ligation, DNA fragments are separated according to their size 
on 0.7 % agarose or 8% acrylamide gels, purified by electrophoresis and 
then electroelution, extracted with phenol, precipitated with ethanol and 
then incubated in a buffer comprising 50 mM Tris-HCl, pH 7.4, 10 mM 
MgCl.sub.2, 10 mM, DTT, 2 mM ATP in the presence of phage T4 DNA ligase 
(Biolabs). Oligo-nucleotides are synthesized using phosphoramidite 
chemistry with the latter derivatives protected at the b position by a 
cyanoethyl group (Sinha et al., 1984, Giles 1985), with the Biosearch 8600 
automatic DNA synthesizer, using the manufacturer's recommendations. 
The ligated DNAs are used to transform the following strains rendered 
competent.dagger.: E.coli MC1060 [(LacIOPZYA)X74, galU, galK, strA.sup.r, 
hsdR] (Casadaban et al., 1983); HB101 [hsdS20, supE44, recA13, ara-14, 
proA2, lacY1, galK2, rpsL20, xyl-5, mtl-1, .lambda.-, F-] (Maniatis et 
al., 1989); and DH5.alpha. [endA1 hsdR17 supE44 thi-1 recA1 gyrA96 relA1 
.lambda.-.PHI.80 dlacZ.DELTA.M15] for the plasmids. 
LB and 2XTY culture media are used for the bacteriological part (Maniatis 
et al., 1989). 
Plasmid DNAs are purified according to the alkaline lysis technique 
(Maniatis et al., 1989). 
Definition of the terms employed and abbreviations. 
Recombinant DNA: set of techniques which make it possible either to 
combine, within the same microorganism, DNA sequences which are not 
naturally combined, or to mutagenize a DNA fragment specifically. 
ATP: adenosine 5'-triphosphate 
BSA: bovine serum albumin 
PBS: 10 mM phosphate buffer, 150 mM NaCl, pH 7.4 
dNTP: 2'-deoxyribonucleoside 5'-triphosphates 
DTT: dithiothreitol 
kb: kilobases 
bp: base pairs 
EXAMPLE 1 
Construction of a Plasmid Carrying the attP and attB Sequences of the 
Bacteriophage, in Repeated Direct Orientations. 
The plasmid pNH16a was used as starting material, inasmuch as it already 
contains a fragment of bacteriophage .lambda. carrying the attP sequence 
(Hasan and Szybalski, 1987). This plasmid was digested with EcoRI. 
Oligonucleotides which contain the attB sequence (Landy, 1989) were 
synthesized. They have the following sequence: 
Oligonucleotide 5476 (SEQ ID No.1) 
5'-AATTGTGAAGCCTGCTTTTTTATACTAACTTGAGCGG-3' 
Oligonucleotide 5477 (SEQ ID No.2) 
5'-AATTCCGCTCAAGTTAGTATAAAAAAGCAGGCTTCAC-3' 
They were hybridized to re-form the attB sequence and then ligated at the 
EcoRI site of the 4.2-kb EcoRI fragment of pNH16a (Hasan and Szybalski, 
1987). After transformation of DH5.alpha., a recombinant clone was 
retained. The plasmid thereby constructed was designated pXL2648 (see FIG. 
4). This plasmid contains the attP and attB sequences of the bacteriophage 
in the direct orientation. Under the action of the integrase of the 
bacteriophage (Int protein), there should be excision of the sequences 
lying between the two att sites. This results in separation of the 
material inserted between the two att sequences from the origin of 
replication and from the resistance marker of the plasmid, which are 
positioned on the outside. 
EXAMPLE 2 
Obtaining a Minicircle in vivo in E.coli. 
A cassette for resistance to kanamycin was cloned at the EcoRI site of 
plasmid pXL2648 (FIG. 4). This cassette originates from the plasmid 
pUC4KIXX (Pharmacia Biotech.). For this purpose, 10 .mu.g of plasmid 
pUC4KIXX were digested with EcoRI and then separated by agarose gel 
electrophoresis; the 1.6-kb fragment containing the kanamycin resistance 
marker was purified by electro-elution; it was then ligated to plasmid 
pXL2648 linearized with EcoRI. The recombinant clones were selected after 
transformation into E.coli DH5.alpha. and selection for resistance to 
kanamycin. The expected restriction profile was observed on one clone; 
this plasmid clone was designated pXL2649 (FIG. 4). This plasmid was 
introduced by transformation into two E.coli strains: 
D1210 [hsdS20, supE44, recA13, ara-14, proA2, lacY1, galK2, rpsL20, xyl-5, 
mtl-1, .lambda..sup.-, F-, lacIg] (Sadler et al., 1980). 
D1210HP, which corresponds to DH1210 lysogenized with the phage xis.sup.- 
(Xis.sup.- Kil.sup.-) cI857 (Podjaska et al., 1985). The D1210HP strain 
[supE44 ara-14 galK2 .DELTA.(gpt-proA)62 rpsL20 xy15 mtl1 recA13 
.DELTA.(mcrC-mrr) hsdS lacl.sup.q ] (.lambda.[cl857 xis.sup.- ki1.sup.- 
]), accession number I-2314, was deposited on Sep. 15, 1999 with the 
Collection National de Cultures de Microorganisms (CNCM), Institut 
Pasteur, 25 rue du Docteur Roux, F-75724 Paris Cedex 15, FRANCE. 
The transformants were selected at 30.degree. C. on 2XTY medium with 
kanamycin (50 mg/l). After reisolation on selective medium, the strains 
were inoculated into 5 ml of L medium supplemented with kanamycin (50 
mg/l). After 16 h of incubation at 30.degree. C. with agitation (5 cm of 
rotational amplitude), the cultures were diluted to 1/100 in 100 ml of the 
same medium. These cultures were incubated under the same conditions until 
an OD.sub.610 of 0.3 was reached. At this point, half of the culture was 
removed and then incubated for 10 min at 42.degree. C. to induce the lytic 
cycle of the phage, hence the expression of the integrase. After this 
incubation, the cultures were transferred again to 30.degree. C. and then 
incubated for 1 h under these conditions. Next, culturing was stopped and 
minipreparations of plasmid DNA were produced. Irrespective of the 
conditions, in the strain D1210, the agarose gel electrophoresis profile 
of the undigested plasmid DNA of plasmid pXL2649 is unchanged, as is also 
the case in the strain D1210HP which has not been thermally induced. On 
the contrary, in D1210HP which has been incubated for 10 min at 42.degree. 
C. and then cultured for 1 hour at 30.degree. C., it is found that there 
is no longer a plasmid, but two circular DNA molecules: one of low 
molecular weight, migrating faster and containing an EcoRI site; and one 
of higher molecular weight, containing a unique BglI site, as expected. 
Hence there has indeed been excision of the sequences present between the 
two att sequences, and generation of a minicircle bereft of any origin of 
replication. This supercoiled circular DNA not carrying an origin of 
replication is termed a minicircle. This name takes, in effect, better 
account of the circular nature of the molecule. The starting plasmid 
pXL2649 is present, but it represents approximately 10% of the plasmid 
which has excised the sequences flanked by att. 
The minicircle may then be purified by standard techniques of plasmid DNA 
purification, since it is supercoiled like plasmid DNA. These techniques 
comprise, inter alia, purification on a cesium chloride density gradient 
in the presence of ethidium bromide, or alternatively the use of anion 
exchange columns (Maniatis et al., 1989). In addition, if the plasmid DNA 
corresponding to the origin of replication and to the selectable marker is 
considered to be present in an excessively large amount, it is always 
possible, after purification, to use one or more restriction enzymes which 
will digest the plasmid and not the minicircle, enabling them to be 
separated by techniques that separate supercoiled DNA from linear DNA, 
such as in a cesium chloride density gradient in the presence of ethidium 
bromide (Maniatis et al., 1989). 
EXAMPLE 3 
Obtaining a Minicircle Containing a Cassette for the Expression of 
Luciferase. 
In order to test the use of these minicircles in vivo, a reporter gene with 
the sequences required for its expression was cloned into plasmid pXL2649 
(see Example 2). This was done using, more especially, a 3150-bp 
BqlII-BamHI cassette originating from pGL2-Control (Promega Biotech). This 
cassette contains the SV40 early promoter, the enhancer of the SV40 early 
promoter, the luciferase gene of Photinus pyralis and a polyadenylation 
site derived from SV40. The 3150-bp BglII-BamHI fragment was cloned at the 
BamHI site of pXL2649 digested with BamHI so as to replace the cassette 
for resistance to kanamycin by the cassette for the expression of 
luciferase from pGL2-control. The plasmid thus constructed was called 
pXL2650. In this plasmid, the attP and attB sites flank the cassette for 
the expression of luciferase. Site-specific recombination enables only the 
sequences required for the expression of luciferase together with the 
luciferase gene to be excised. This recombination may be carried out 
exactly as described in Example 2. A minicircle such as plasmid pXL2650 
may be used thereafter in in vivo or in vitro transfection experiments. 
A 1-liter culture of the strain D1210HP pXL2650 in 2XTY medium supplemented 
with ampicillin (50 mg/ml) was set up at 30.degree. C. At an OD.sub.610 
equal to 0.3, the culture was transferred to 42.degree. C. for 20 min, 
then replaced for 20 min at 30.degree. C. The episomal DNA was prepared by 
the clear lysate technique (Maniatis et al., 1989), followed by a cesium 
chloride density gradient supplemented with ethidium bromide (Maniatis et 
al., 1989), then by an extraction of the ethidium bromide with isopropanol 
and by a dialysis. This DNA was shown to contain the minicircle. 100 .mu.g 
of this preparation were digested with PstI, and the hydrolysate was then 
subjected to a cesium chloride density gradient supplemented with ethidium 
bromide (Maniatis et al., 1989). An identical result is obtained when the 
preparation is digested jointly with AlwNI and XmnI. The supercoiled form 
was recovered and, after removal of the ethidium bromide (Maniatis et 
al.), it was found to correspond only to the minicircle, lacking an origin 
of replication and any marker gene. This minicircle preparation may be 
used for in vitro and in vivo transfection experiments. 
EXAMPLE 4 
In vitro Transfection of Mammalian Cells, and More Especially of Human 
Cells, with a Minicircle. 
The minicircle DNA containing the luciferase gene of Photinus pyralis as 
described in Example 3, that is to say corresponding to the minicircle 
generated from plasmid pXL2650, is diluted in 150 mM NaCl and mixed with a 
transfectant. It is possible to use various commercial transfectants, such 
as dioctadecylamidoglycylspermine (DOGS, Transfectam.TM., Promega), 
Lipofectin.TM. (Gibco-BRL), and the like, in different positive/negative 
charge ratios. By way of illustration, the transfecting agent was used in 
charge ratios greater than or equal to 3. The mixture is vortexed, left 
for 10 minutes at room temperature, diluted in culture medium without 
fetal calf serum, and then added to the cells in the proportion of 2 .mu.g 
of DNA per culture well. The cells used are Caco-2, derived from a human 
colon adenocarcinoma, cultured according to a protocol described (Wils et 
al., 1994) and inoculated on the day before the experiment into 48-well 
culture plates in the proportion of 50,000 cells/well. After two hours at 
37.degree. C., 10% v/v of fetal calf serum is added and the cells are 
incubated for 24 hours at 37.degree. C. in the presence of 5% CO.sub.2. 
The cells are washed twice with PBS and the luciferase activity is 
measured according to the protocol described (such as the Promega kit). It 
is possible to use other lines (fibroblasts, lymphocytes, etc.) 
originating from different species, or alternatively cells taken from an 
individual (fibroblasts, keratinocytes, lymphocytes, etc.) and which will 
be reinjected into him or her after transfection. 
EXAMPLE 5 
In vitro Transfection of NIH 3T3 Cells. 
The minicircle DNA containing the luciferase gene of Photinus pyralis, as 
described in Example 3, that is to say corresponding to the minicircle 
generated from plasmid pXL2650, was transfected in vitro into mammalian 
cells; pXL2650 and PGL2-Control (Promega Biotech.), which contain the same 
expression cassette, were used as control. The cells used are NIH 3T3 
mouse fibroblasts, inoculated on the day before the experiment into 
24-well culture plates in the proportion of 50,000 cells per well. The 
plasmid is diluted in 150 mM NaCl and mixed with the lipofectant 
RPR115335. However, it is possible to use various other commercial agents 
such as dioctadecylaminoglycylspermine (DOGS, Transfectam.TM., Promega) 
(Demeneix et al., Int. J. Dev. Biol. 35 (1991) 481), Lipofectin.TM. 
(Gibco-BRL) (Fegner et al., Proc. Natl. Acad. Sci. USA 84 (1987) 7413), 
and the like. A positive charge of the lipofectant/negative charge of the 
DNA ratio equal to or greater than 3 is used. The mixture is vortexed, 
left for ten minutes at room temperature, diluted in medium without fetal 
calf serum, and then added to the cells in the proportion of 0.5 mg of DNA 
per culture well. After two hours at 37.degree. C., 10% by volume of fetal 
calf serum is added and the cells are incubated for 48 hours at 37.degree. 
C. in the presence of 5% CO.sub.2. The cells are washed twice with PBS and 
the luciferase activity is measured according to the protocol described 
(Promega kit, Promega Corp. Madison, Wis.), on a Lumat LB9501 luminometer 
(EG and G Berthold, Evry). The transfection results corresponding to the 
conditions which have just been stated are presented in FIG. 5. They show 
unambiguously that the minicircle has the same transfection properties as 
plasmids possessing an origin of replication. Thus these minicircles could 
be used in the same way as standard plasmids in gene therapy applications. 
EXAMPLE 6 
Affinity Purification of a Minicircle Using a Triple-helix Interaction. 
This example describes a method of purification of a minicircle according 
to the invention from a mixture containing the plasmid form which has 
excised it, by triple-helix type interactions which will take place with a 
synthetic DNA sequence carried by the minicircle to be purified. This 
example demonstrates how the technology of purification by triple-helix 
formation may be used to separate a minicircle from a plasmid form which 
has excised it. 
6-1. Obtaining a Minicircle Containing a Synthetic 
Homopurine-homopyrimidine Sequence 
6-1.1. Insertion of a homopurine-homopyrimidine sequence into plasmid 
pXL2650 
Plasmid pXL2650 possesses a unique BamHI site immediately after the 
cassette containing the luciferase gene of Photinus pyralis. This unique 
site was used to clone the following two oligonucleotides: 
4957 (SEQ ID No.3) 
5'-GATCCGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAC-3' 
4958 (SEQ ID No.4) 
5'-GATCGTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCG-3' 
These oligonucleotides, when hybridized and cloned into plasmid pXL2650, 
introduce a homopurine-homopyrimidine sequence (GAA).sub.17, as described 
above. 
To carry out this cloning, the oligonucleotides were first hybridized in 
the following manner. One .mu.g of each of these two oligonucleotides were 
placed together in 40 ml of a final buffer comprising 50 mM Tris-HCl, pH 
7.4, 10 mM MgCl.sub.2. This mixture was heated to 95.degree. C. and was 
then placed at room temperature so that the temperature would fall slowly. 
Ten ng of the mixture of hybridized oligonucleotides were ligated with 200 
ng of plasmid pXL2650 linearized with BamHI, 30 ml of final. After 
ligation, an aliquot was transformed into DH5. The transformation mixtures 
were plated out on L medium supplemented with ampicillin (50 mg/l). 
Twenty-four clones were digested with PflMI and BamHI. One clone was found 
which had the size of the 950-bp PflMI-BamHI fragment increased by 50 bp. 
This clone was selected and designated pXL2651. 
Plasmid pXL2651 was purified according to the Wizard Megaprep kit (Promega 
Corp., Madison, Wis.) according to the supplier's recommendations. 
6-1.2. Insertion of a homopurine-homopyrimidine sequence into plasmid 
pXL2649 
a) Insertion of new restriction sites on each side of the kanamycin 
cassette of pXL2649. 
Plasmid pXL2649, as described in Example 2, was digested with EcoRI so as 
to take out the kanamycin cassette originating from plasmid pUC4KIXX 
(PharmaciaBiotech, Uppsala, Sweden). For this purpose, 5 mg of plasmid 
pXL2649 were digested with EcoRI. The 4.2-kb fragment was separated by 
agarose gel electrophoresis and purified by electroelution. 
In addition, the plasmid pXL1571 was used. The latter was constructed from 
the plasmid pFR10 (Gene 25 (1983), 71-88), into which the 1.6-kb fragment 
originating from pUC4KIXX, corresponding to the kanamycin gene, was 
inserted at the SstI site. This cloning enabled 12 new restriction sites 
to be inserted on each side of the kanamycin gene. 
Five micrograms of pXL1571 were dialysed with EcoRI. The 1.6-kb fragment 
corresponding to the kanamycin gene was separated by agarose gel 
electrophoresis and purified by electroelution. It was then ligated with 
the 4.2-kb EcoRI fragment of pXL2649. The recombinant clones were selected 
after transformation into E. coli DH5a and selection for resistance to 
kanamycin and to ampicillin. The expected restriction profile was observed 
on one clone; this plasmid clone was designated pXL2791. 
b) Extraction of the kanamycin cassette from plasmid pXL2791 
Plasmid pXL2791 was digested with SstI so as to take out the kanamycin 
cassette. The 4.2-kb fragment was separated by agarose gel electrophoresis 
and purified with the Jetsorb extraction gel kit (Genomed). It was then 
ligated. The recombinant clones were selected for resistance to ampicillin 
after transformation into E. coli DH5a. The expected restriction profile 
was observed on one clone. This plasmid clone was designated pXL2792. This 
clone comprises, inter alia, SalI and XmaI restriction sites between the 
attP and attB sites. c) Cloning of a homopurine-homopyrimidine sequence as 
well as of a cassette permitting the expression of luciferase between the 
two attP and attB sites of plasmid pXL2792 
Plasmid pXL2727 was used. This plasmid, digested with XmaI and SalI, 
enables a fragment comprising the following to be taken out: the pCMV 
promoter, the luciferase gene of Photinus pyralis, a polyadenylation site 
derived from SV40 and a homopurine-homopyrimidine sequence. The latter was 
obtained after hybridization and cloning of the following two 
oligonucleotides: 
6006: (SEQ ID No.16) 
5'-GATCTGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAACTGCAGATCT-3' 
6008: (SEQ ID No.17) 5'-GATCAGATCTGCAGTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCT 
TCTTCTTCTTCTTCTTCA-3' 
The homopurine-homopyrimidine sequence present in pXL2727 was sequenced by 
the Sequenase Version 2.0 method (United States Biochemical Corporation). 
The result obtained shows that the homopurine-homopyrimidine sequence 
actually present in plasmid pXL2727 contains 10 repeats (GAA-CTT), and not 
17 as the sequence of the oligonucleotides 6006 and 6008 suggested would 
be the case. The sequence actually present in plasmid pXL2727, read after 
sequencing on the strand corresponding to the oligonucleotide 6008, is as 
follows: 
5'-GATCAGATCTGCAGTCTCTTCTTCTTCTTCTTCTTCTTCTTCT TCTTCTCTTCTCA-3' (SEQ ID 
No.18) 
One microgram of pXL2727 was digested with XmaI and SalI. The 3.7-kb 
fragment was separated by agarose gel electrophoresis and purified with 
the Jetsorb extraction gel kit (Genomed). In addition, 1.7 mg of pXL2792 
were digested with XmaI and SalI. The 4.2-kb fragment was separated on 
agarose gel, purified with the Jetsorb extraction gel kit (Genomed) and 
ligated with the 3.7-kb XmaI-SalI fragment of pXL2727. The recombinant 
clones were selected after transformation into E. coli DH5a and selection 
for resistance to ampicillin. The expected restriction profile was 
observed on one clone; this clone was designated pXL2793. Plasmid pXL2793 
was purified using a caesium chloride density gradient according to a 
method already described (Maniatis et al., 1989). 
6-2. Preparation of the Column Enabling Triple-helix Type Interactions with 
a Homopurine-homopyrimidine Sequence Present in the Minicircle to be 
Effected 
The column was prepared in the following manner: 
The column used is a 1-ml HiTrap column activated with NHS 
(N-hydroxysuccinimide, Pharmacia), connected to a peristaltic pump (flow 
rate&lt;1 ml/min). The specific oligonucleotide used possesses an NH.sub.2 
group at the 5' end. 
For plasmid pXL2651, its sequence is as follows: 
5'-GAGGCTTCTTCTTCTTCTTCTTCTT-3' (SEQ ID No.5) 
For plasmid pXL2793, its sequence is as follows (oligo 116418): 
5'-CTTCTTCTTCTTCTTCTTCTT-3' (SEQ ID No. 19) 
The buffers used are the following: 
Coupling buffer: 0.2 M NaHCO.sub.3, 0.5 M NaCl, pH 8.3. 
Washing buffer: 
Buffer A: 0.5 M ethanolamine, 0.5 M NaCl, pH 8.3. 
Buffer B: 0.1 M acetate, 0.5 M NaCl, pH 4. 
Fixing and eluting buffer: 
Buffer F: 2 M NaCl, 0.2 M acetate, pH 4.5. 
Buffer E: 1 M Tris-HCl, pH 9, 0.5 mM EDTA. 
The column is prepared in the following manner: 
The column is washed with 6 ml of 1 mM HCl, and the oligonucleotide diluted 
in the coupling buffer (50 nmol in 1 ml) is then applied to the column and 
left for 30 minutes at room temperature. The column is washed with 3 ml of 
coupling buffer, then with 6 ml of buffer A, followed by 6 ml of buffer B. 
The latter two buffers are applied three times in succession to the 
column. In this way, the oligonucleotide is linked covalently to the 
column via a CONH link. The column is stored at 4.degree. C. in PBS, 0.1% 
NaN.sub.3. 
6-3. Purification of a Minicircle Containing a Synthetic 
Homopurine-homopyrimidine Sequence, by a Triple-helix Type Interaction 
6-3.1. Purification of plasmid pXL2651 
Plasmid pXL2651 was introduced into the strain D1210HP. This recombinant 
strain [D1210HP (pXL2651)] was cultured as described in Example 3 so as to 
generate the minicircle containing the luciferase gene of Photinus 
pyralis. Twenty ml of culture were removed and centrifuged. The cell 
pellet is taken up in 1.5 ml of 50 mM glucose, 25 mM Tris-HCl, pH 8, 10 mM 
EDTA. Lysis is carried out with 2 ml of 0.2 M NaOH, 1% SDS, and 
neutralization with 1.5 ml of 3 M potassium acetate, pH 5. The DNA is then 
precipitated with 3 ml of 2-propranol, and the pellet is taken up in 0.5 
ml of 0.2 M sodium acetate, pH 5, 0.1 M NaCl and loaded onto an 
oligonucleotide column capable of forming triple-helix type interactions 
with poly(GAA) sequences contained in the minicircle, as described above. 
After the column has been washed beforehand with 6 ml of buffer F, the 
solution containing the minicircle to be purified is incubated, after 
being applied to the column, for two hours at room temperature. The column 
is washed with 10 ml of buffer F and elution is then carried out with 
buffer E. 
Purified DNA corresponding to the minicircle is thereby obtained. The 
minicircle obtained, analysed by agarose gel electrophoresis and ethidium 
bromide staining, takes the form of a single band of supercoiled circular 
DNA. Less than 5% of starting plasmid pXL2651 is left in the preparation. 
6-3.2. Purification of plasmid pXL2793 
The 7.9-kb plasmid pXL2793 was introduced into the strain D1210HP. This 
recombinant strain was cultured as described in Example 3, so as to 
generate the 4-kb minicircle containing the luciferase gene of Photinus 
pyralis and a 3.9-kb plasmid. Two hundred ml of culture were removed and 
centrifuged. The cell pellet was treated with the Wizard Megaprep kit 
(Promega Corp., Madison, Wis.) according to the supplier's 
recommendations. The DNA was taken up in a final volume of 2 ml of 1 mM 
Tris, 1 mM EDTA, pH 8. Two hundred and fifty microliters of this plasmid 
sample were diluted with buffer F in a final volume of 2.5 ml. The column 
was washed beforehand with 6 ml of buffer F. The whole of the diluted 
sample was loaded onto an oligonucleotide column capable of forming 
triple-helix type interactions with poly(GAA) sequences contained in the 
minicircle, prepared as described above. After washing with 10 ml of 
buffer F, elution is carried out with buffer E. The eluted sample is 
recovered in 1-ml fractions. 
By this method, purified DNA corresponding to the minicircle generated from 
pXL2793 is obtained. The DNA sample eluted from the column was analysed by 
agarose gel electrophoresis and ethidium bromide staining, and by enzyme 
restriction. For this purpose, the eluted fractions which were shown to 
contain DNA by assay at OD.sub.260 nm were dialysed for 24 hours against 1 
mM Tris, 1 mM EDTA, then precipitated with isopropanol and taken up in 200 
ml of H.sub.2 O. Fifteen microliters of the sample thereby obtained were 
digested with SalI, this restriction site being present in the minicircle 
and not in the 3.9-kb plasmid generated by the recombination, or with 
XmnI, this restriction site being present in the 3.9-kb plasmid generated 
by the recombination and not in the minicircle. The result obtained is 
presented in FIG. 7, showing that the minicircle has been purified of the 
recombinant plasmid. 
EXAMPLE 7 
In vivo Transfection of Mammalian Cells with a Minicircle 
This example describes the transfer of a minicircle coding for the 
luciferase gene into the brain of newborn mice. The minicircle (30 .mu.g) 
is diluted in sterile 150 mM NaCl to a concentration of 1 .mu.g/.mu.l. A 
synthetic transfectant such as dioctadecylamidoglycylspermine (DOGS) is 
then added in a positive/negative charge ratio less than or equal to 2. 
The mixture is vortexed, and 2 .mu.g of DNA are injected into the cerebral 
cortex of anaesthetized newborn mice using a micromanipulator and a 
microsyringe. The brains are removed 48 hours later, homogenized and 
centrifuged and the supernatant is used for the assay of luciferase by the 
protocols described (such as the Promega kit). 
EXAMPLE 8 
Use of the par Locus of RK2 to Reduce the Presence of Minicircle or 
Miniplasmid Topoisomers 
This example demonstrates the presence of topological forms derived i) from 
the plasmid possessing the attP and attB sequences in the direct 
orientation, ii) from the minicircle or iii) from the miniplasmid, after 
the action of the integrase of bacteriophage l in E. coli. This example 
also shows that these topological or oligomeric forms may be resolved by 
using the par locus of RK2 (Gerlitz et al., 1990 J. Bacteriol. 172 p. 
6194). In effect, this locus contains, in particular, the parA gene coding 
for a resolvase acting at the mrs (multimer resolution system) site (Eberl 
et al., 1994 Mol. Microbiol. 12 p. 131). 
8-1. Construction of Plasmids pXL2777 and pXL2960 
Plasmids pXL2777 and pXL2960 are derived from the vector pXL2776, and 
possess in common the minimal replicon of ColE1, the gene of the 
transposon Tn5 coding for resistance to kanamycin and the attP and attB 
sequences of bacteriophage l in the direct orientation. These plasmids 
differ in respect of the genes inserted between the attP and attB 
sequences, in particular pXL2777 contains the omegon cassette (coding for 
the gene for resistance to spectinomycin) whereas plasmid pXL2960 carries 
par locus of RK2. 
8-1.1. Minimal vector pXL2658 
The vector pXL2658 (2.513 kb) possesses the minimal replicon of ColE1 
originating from pBluescript (ori) and the gene of the transposon Tn5 
coding for resistance to kanamycin (Km) as selectable marker. After the 
BsaI end has been blunted by the action of the Klenow enzyme, the 1.15-kb 
BsaI-PvuII fragment of pBKS+ (obtained from Stratagene) was cloned with 
the 1.2-kb SmaI fragment of pUC4KIXX (obtained from Pharmacia) to generate 
the plasmid pXL2647. The oligo-nucleotides 5542 5'(AGC TTC TCG AGC TGC AGG 
ATA TCG AAT TCG GAT CCT CTA GAG CGG CCG CGA GCT CC)3' (SEQ ID No.20) and 
5543 5'(AGC TGG AGC TCG CGG CCG CTC TAG AGG ATC CGA ATT CGA TAT CCT GCA 
GCT CGA GA)3' (SEQ ID No.21) were hybridized with one another and then 
cloned at the HindIII site of pXL2647; in this way pXL2658 is constructed. 
In this plasmid, the multiple cloning site is SstI, NotI, XbaI, BamHI, 
EcoRI, EcoRV, PstI, XhoI and HindIII between the origin of replication and 
the gene coding for resistance to kanamycin. 
8-1.2. Vector pXL2776 containing the attP and attB sequences of phage l 
The vector pXL2776 (2.93 kb) possesses the minimal replicon of ColE1 
originating from pBluescript, the gene coding for resistance to kanamycin 
and the attP and attB sequences of bacteriophage l in the direct 
orientation, see FIG. 8. The 29-bp attB sequence (Mizuuchi et al., 1980 
Proc. Natl. Acad. Sci. USA 77 p. 3220) was introduced between the SacI and 
HindIII restriction sites of pXL2658 after the sense oligonucleotide 6194 
5'(ACT AGT GGC CAT GCA TCC GCT CAA GTT AGT ATA AAA AAG CAG GCT TCA G)3' 
(SEQ ID No.22) has been hybridized with the antisense oligonucleotide 6195 
5'(AGC TCT GAA GCC TGC TTT TTT ATA CTA ACT TGA GCG GAT GCA TGG CCA CTA GTA 
GCT)3' (SEQ ID No.23) in such a way that the SacI and HindIII sites are no 
longer re-formed after cloning. This plasmid, the sequence of which was 
verified with respect to attB, is then digested with SpeI and NsiI in 
order to introduce in it the attP sequence flanked by the NsiI and XbaI 
restriction sites and thus to generate plasmid pXL2776. The attP sequence 
was obtained by PCR amplification using plasmid pXL2649 (described in 
Example 2) as template, the sense oligonucleotide 6190 5'(GCG TCT AGA ACA 
GTA TCG TGA TGA CAG AG)3' (SEQ ID No.24) and the antisense oligonucleotide 
6191 5'(GCC AAG CTT AGC TTT GCA CTG GAT TGC GA)3' (SEQ ID No.25), and 
performing 30 cycles during which the hybridization temperature is 
50.degree. C. The PCR product digested at the XbaI and HindIII sites was 
cloned into the phage M13mpEH between the XbaI and HindIII sites. The 
amplified sequence is identical to the attP sequence described in Lambda 
II (edited by R. W. Hendrix, J. W. Roberts, F. W. Stahl, R. A. Weisberg; 
Cold Spring Harbor Laboratory 1983) between positions 27480 and 27863. 
8-1.3. Plasmid pXL2777 
Plasmid pXL2777 (6.9 kb) possesses the minimal replicon of ColE1 
originating from pBluescript, the gene coding for resistance to kanamycin, 
the attP and attB sequences of bacteriophage l in the direct orientation 
and separated by the sacB gene coding for levansucrase of B. subtilis (P. 
Gay et al., 1983 J. Bacteriol. 153 p. 1424), and the Sp omegon coding for 
the gene for resistance to spectinomycin Sp and streptomycin Sm (P. 
Prentki et al., 1984 Gene 29 p. 303). The sacB-Sp cassette having EcoRV 
and NsiI cloning ends comes from the plasmid pXL2757 (FR95/01632) and was 
cloned between the EcoRV and NsiI sites of pXL2776 to form pXL2777. 
8-1.4. Plasmid pXL2960 
Plasmid pXL2960 (7.3 kb) possesses the minimal replicon of ColE1 
originating from pBluescript, the gene coding for resistance to kanamycin 
and the attP and attB sequences of bacteriophage l in the direct 
orientation and separated by i) the sacB gene coding for levansucrase of 
B. subtilis (P. Gay et al., 1983 J. Bacteriol. 153 p. 1424) and ii) the 
par locus of RK2 (Gerlitz et al., 1990 J. Bacteriol. 172 p. 6194). The par 
cassette having BamHI ends comes from the plasmid pXL2433 (PCT/FR95/01178) 
and was introduced between the BamHI sites of pXL2777 to generate pXL2960. 
8-2. Resolution of Minicircle or Miniplasmid Topoisomers 
Plasmids pXL2777 and pXL2960 were introduced by transformation into E. coli 
strain D1210HP. The transformants were selected and analysed as described 
in Example 2, with the following modifications: the expression of the 
integrase was induced at 42.degree. C. for 15 min when the optical density 
of the cells at 610 nm is 1.8, and the cells are then incubated at 
30.degree. C. for 30 min, see FIG. 9, or for a period varying from 2 
minutes to 14 hours (O/N), see FIG. 10. The plasmid DNA originating from 
uninduced and induced cultures was then analysed on agarose gel before or 
after digestion with a restriction enzyme exclusive to the minicircle 
portion (EcoRI) or miniplasmid portion (BglII), see Figure Y, or after the 
action of DNA topoisomerase A or the gyrase of E. coli. The supercoiled 
dimer forms of minicircle or miniplasmid are clearly revealed by i) their 
molecular weight, ii) their linearization by the restriction enzyme, iii) 
their change in topology through the action of topoisomerase A (relaxed 
dimer) or of the gyrase (supersupercoiled dimer), iv) specific 
hybridization with an internal fragment peculiar to the minicircle or the 
miniplasmid. Other topological forms of higher molecular weights than that 
of the initial plasmid originate from the initial plasmid or the 
minicircle or the miniplasmid, since they disappear after digestion with 
the restriction enzyme exclusive to the minicircle portion (EcoRI) or 
miniplasmid portion (BglII). These forms are much less abundant with 
pXL2960 than with pXL2777 as initial plasmid, see FIG. 10. In particular, 
the dimer form of minicircle is present to a not insignificant extent with 
plasmid pXL2777, whereas it is invisible with plasmid pXL2960 when the 
cells are incubated for at least 30 min at 30.degree. C., see FIGS. 9 and 
10. It should be noted that minicircle dimers are observed at the 
beginning of the kinetic experiment with pXL2960 (2 to 10 min), and are 
thereafter resolved (after 30 min), see FIG. 10. Consequently, the par 
locus leads to a significant reduction in the oligomeric/topological forms 
resulting from the action of the integrase of bacteriophage l in E. coli 
on plasmids containing the attP and attB sequences in the direct 
orientation. 
IDENTIFICATION OF THE NUCLEOTIDE SEQUENCES 
SEQ ID No.1: oligonucleotide 5476: 
5'-AATTGTGAAGCCTGCTTTTTTATACTAACTTGAGCGG-3' 
SEQ ID No.2: oligonucleotide 5477 
5'-AATTCCGCTCAAGTTAGTATAAAAAAGCAGGCTTCAC-3' 
SEQ ID No.3: oligonucleotide 4957: 
5'-GATCCGAAGAGAGAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAC-3' 
SEQ ID No.4: oligonucleotide 4958: 
5'-GATCGTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCG-3' 
SEQ ID No.5: oligonucleotide poly-CTT: 5'-GAGGCTTCTTCTTCTTCTTCTTCTT-3' 
SEQ ID No.6: (attB sequence of phage lambda): 5'-CTGCTTTTTTATACTAACTTG-3' 
SEQ ID No.7: (attP sequence of phage lambda): 5'-CAGCTTTTTTATACTAAGTTG-3' 
SEQ ID No.8: (attB sequence of phage P22): 5'-CAGCGCATTCGTAATGCGAAG-3' 
SEQ ID No.9: (attP sequence of phage P22): 5'-CTTATAATTCGTAATGCGAAG-3' 
SEQ ID No.10: (attB sequence of phage F80): 5'-AACACTTTCTTAAATGGTT-3' 
SEQ ID No.11: (attP sequence of phage F80): 5'-AACACTTTCTTAAATTGTC-3' 
SEQ ID No.12: (attB sequence of phage HP1): 5'-AAGGGATTTAAAATCCCTC-3' 
SEQ ID No.13: (attP sequence of phage HP1): 5'-ATGGTATTTAAAATCCCTC-3' 
SEQ ID No.14: (att sequence of plasmid pSAM2): 
5'-TTCTCTGTCGGGGTGGCGGGATTTGAACCCACGACCTCTTCGTCCCGAA-3' 
SEQ ID No.15: (Recognition sequence of the resolvase of the transposon 
Tn3): 5'-CGTCGAAATATTATAAATTATCAGACA-3' 
SEQ ID No.16: oligonucleotide 6006: 
5'-GATCTGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAACTGCAGATCT-3' 
SEQ ID No.17: oligonucleotide 6008: 
5'-GATCAGATCTGCAGTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCA-3' 
SEQ ID No.18: (Sequence present in plasmid pXL2727 corresponding to the 
oligonucleotide 6008): 
5'-GATCAGATCTGCAGTCTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTCTTCTTCA-3' 
SEQ ID No.19: (oligonucleotide 116418): 5'-CTTCTTCTTCTTCTTCTTCTT-3' 
SEQ ID No.20: (oligonucleotide 5542): 
5'-AGCTTCTCGAGCTGCAGGATATCGAATTCGGATCCTCTAGAGCGGCCGCGAGCTCC-3' 
SEQ ID No.21: (oligonucleotide 5543): 
5'-AGCTGGAGCTCGCGGCCGCTCTAGAGGATCCGAATTCGATATCCTGCAGCTCGAGA-3' 
SEQ ID No.22: sense oligonucleotide 6194: 
5'-ACTAGTGGCCATGCATCCGCTCAAGTTAGTATAAAAAAGCAGGCTTCAG-3' 
SEQ ID No.23: antisense oligonucleotide 6195: 
5'-AGCTCTGAAGCCTGCTTTTTTATACTAACTTGAGCGGATGCATGGCCACTAGTAGCT-3' 
SEQ ID No.24: sense oligonucleotide 6190: 
5'-GCGTCTAGAACAGTATCGTGATGACAGAG-3' 
SEQ ID No.25: antisense oligonucleotide 6191: 
5'-GCCAAGCTTAGCTTTGCACTGGATTGCGA-3' 
Bibliographic References 
Ausubel et al. Current protocols in molecular biology 1987-1988. John 
Willey and Sons, New York. 
Behr J. P. 1993. Acc. Chem. Res. 26:274-278. 
Casadaban et al. 1983. Methods Enzymol. 100, 293-308. 
Cotten et al. E. 1993. Curr. Biol. 4:705-710. 
Giles, J. W. 1985. Am. Biotechnol., November/December. 
Hasan et al. 1987. Gene 56:145-151. 
Jain, R. K. 1987. Cancer Res. 47:3039-3051. 
Landford et al. 1986. Cell 46:575-582. 
Landy, A. 1989. Ann. Rev. Biochem. 58:913-949. 
Maniatis, T., E. F. Fritsch, and J. Sambrook. 1989. Molecular cloning: a 
laboratory manual, second edition. Cold Spring Harbor Laboratory, Cold 
Spring Harbor Laboratory Press, New York. 
Nabel et al. 1992. Human Gene Therapy 3:399-410. 
Podhajska et al. 1985. Gene 40:163:168. 
Sadler et al. 1980. Gene, 8:279-300. 
Sinha et al. 1984. Acids Res., 12, 4539-4557. 
Stark et al. 1992. Trends Genet. 8:432-439. 
Viera et al. 1982. Gene, 19, 259-268. 
Wils et al. Biochem. Pharmacol. 48:1528-1530. 
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# SEQUENCE LISTING 
- - - - (1) GENERAL INFORMATION: 
- - (iii) NUMBER OF SEQUENCES: 25 
- - - - (2) INFORMATION FOR SEQ ID NO:1: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 37 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "Oligonucleotide" 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
- - AATTGTGAAG CCTGCTTTTT TATACTAACT TGAGCGG - # 
- # 37 
- - - - (2) INFORMATION FOR SEQ ID NO:2: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 37 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "Oligonucleotide" 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
- - AATTCCGCTC AAGTTAGTAT AAAAAAGCAG GCTTCAC - # 
- # 37 
- - - - (2) INFORMATION FOR SEQ ID NO:3: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 57 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "Oligonucleotide" 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
- - GATCCGAAGA AGAAGAAGAA GAAGAAGAAG AAGAAGAAGA AGAAGAAGAA GA - #AGAAC 
57 
- - - - (2) INFORMATION FOR SEQ ID NO:4: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 57 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "Oligonucleotide" 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
- - GATCGTTCTT CTTCTTCTTC TTCTTCTTCT TCTTCTTCTT CTTCTTCTTC TT - #CTTCG 
57 
- - - - (2) INFORMATION FOR SEQ ID NO:5: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 25 base - #pairs 
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(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "Oligonucleotide" 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
- - GAGGCTTCTT CTTCTTCTTC TTCTT - # - # 
25 
- - - - (2) INFORMATION FOR SEQ ID NO:6: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "Oligonucleotide" 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
- - CTGCTTTTTT ATACTAACTT G - # - # 
- #21 
- - - - (2) INFORMATION FOR SEQ ID NO:7: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base - #pairs 
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(A) DESCRIPTION: /desc - #= "Oligonucleotide" 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
- - CAGCTTTTTT ATACTAAGTT G - # - # 
- #21 
- - - - (2) INFORMATION FOR SEQ ID NO:8: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "Oligonucleotide" 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
- - CAGCGCATTC GTAATGCGAA G - # - # 
- #21 
- - - - (2) INFORMATION FOR SEQ ID NO:9: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "Oligonucleotide" 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
- - CTTATAATTC GTAATGCGAA G - # - # 
- #21 
- - - - (2) INFORMATION FOR SEQ ID NO:10: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 19 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "Oligonucleotide" 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
- - AACACTTTCT TAAATGGTT - # - # 
- # 19 
- - - - (2) INFORMATION FOR SEQ ID NO:11: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 19 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "Oligonucleotide" 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
- - AACACTTTCT TAAATTGTC - # - # 
- # 19 
- - - - (2) INFORMATION FOR SEQ ID NO:12: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 19 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "Oligonucleotide" 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: 
- - AAGGGATTTA AAATCCCTC - # - # 
- # 19 
- - - - (2) INFORMATION FOR SEQ ID NO:13: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 19 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "Oligonucleotide" 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: 
- - ATGGTATTTA AAATCCCTC - # - # 
- # 19 
- - - - (2) INFORMATION FOR SEQ ID NO:14: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 49 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "Oligonucleotide" 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: 
- - TTCTCTGTCG GGGTGGCGGG ATTTGAACCC ACGACCTCTT CGTCCCGAA - # 
49 
- - - - (2) INFORMATION FOR SEQ ID NO:15: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 27 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "Oligonucleotide" 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: 
- - CGTCGAAATA TTATAAATTA TCAGACA - # - # 
27 
- - - - (2) INFORMATION FOR SEQ ID NO:16: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 66 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "Oligonucleotide" 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: 
- - GATCTGAAGA AGAAGAAGAA GAAGAAGAAG AAGAAGAAGA AGAAGAAGAA GA - 
#AGAACTGC 60 
- - AGATCT - # - # - 
# 66 
- - - - (2) INFORMATION FOR SEQ ID NO:17: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 66 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "Oligonucleotide" 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: 
- - GATCAGATCT GCAGTTCTTC TTCTTCTTCT TCTTCTTCTT CTTCTTCTTC TT - 
#CTTCTTCT 60 
- - TCTTCA - # - # - 
# 66 
- - - - (2) INFORMATION FOR SEQ ID NO:18: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 57 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "Oligonucleotide" 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: 
- - GATCAGATCT GCAGTCTCTT CTTCTTCTTC TTCTTCTTCT TCTTCTTCTC TT - #CTTCA 
57 
- - - - (2) INFORMATION FOR SEQ ID NO:19: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "Oligonucleotide" 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: 
- - CTTCTTCTTC TTCTTCTTCT T - # - # 
- #21 
- - - - (2) INFORMATION FOR SEQ ID NO:20: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 56 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "Oligonucleotide" 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: 
- - AGCTTCTCGA GCTGCAGGAT ATCGAATTCG GATCCTCTAG AGCGGCCGCG AG - #CTCC 
56 
- - - - (2) INFORMATION FOR SEQ ID NO:21: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 56 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "Oligonucleotide" 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: 
- - AGCTGGAGCT CGCGGCCGCT CTAGAGGATC CGAATTCGAT ATCCTGCAGC TC - #GAGA 
56 
- - - - (2) INFORMATION FOR SEQ ID NO:22: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 49 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "Oligonucleotide" 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: 
- - ACTAGTGGCC ATGCATCCGC TCAAGTTAGT ATAAAAAAGC AGGCTTCAG - # 
49 
- - - - (2) INFORMATION FOR SEQ ID NO:23: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 57 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "Oligonucleotide" 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: 
- - AGCTCTGAAG CCTGCTTTTT TATACTAACT TGAGCGGATG CATGGCCACT AG - #TAGCT 
57 
- - - - (2) INFORMATION FOR SEQ ID NO:24: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 29 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "Oligonucleotide" 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: 
- - GCGTCTAGAA CAGTATCGTG ATGACAGAG - # - # 
29 
- - - - (2) INFORMATION FOR SEQ ID NO:25: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 29 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "Oligonucleotide" 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: 
- - GCCAAGCTTA GCTTTGCACT GGATTGCGA - # - # 
29 
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