EP0944717A1 - Method for producing recombinant adenovirus - Google Patents

Abstract

The invention concerns a method for producing recombinant adenovirus by which viral DNA is introduced in a packaging cell culture and the viruses produced are harvested after liberation in the supernatant. The invention also concerns the viruses produced and their use.

Description

 PROCESS FOR PRODUCING RECOMBINANT ADENOVIRUSES

The present invention relates to a new process for the production of recombinant adenoviruses. It also relates to purified viral preparations produced by this process.

Adenoviruses have certain properties which are particularly advantageous for use as a vector for gene transfer in gene therapy. In particular, they have a fairly broad host spectrum, are capable of infecting quiescent cells, do not integrate into the genome of the infected cell, and have not been associated to date with significant pathologies in man. Adenoviruses have thus been used to transfer genes of interest into the muscle (Ragot et al., Nature 361 (1993) 647), the liver (Jaffe et al., Nature genetics 1 (1992) 372), the nervous system (Akli et al., Nature genetics 3 (1993) 224), etc.

Adenoviruses are linear double-stranded DNA viruses approximately 36 (kilobases) kb in size. Their genome includes in particular a repeated inverted sequence (ITR) at each end, an encapsidation sequence (Psi), early genes and late genes. The main early genes are contained in the E1, E2, E3 and E4 regions. Among these, the genes contained in the El region in particular are necessary for viral propagation. The main late genes are contained in regions L1 to L5. The genome of the Ad5 adenovirus has been fully sequenced and is accessible on the database (see in particular Genebank M73260). Likewise, parts or even all of other adenoviral genomes (Ad2, Ad7, Adl2, etc.) have also been sequenced.

For their use in gene therapy, different vectors derived from adenoviruses have been prepared, incorporating different therapeutic genes. In each of these constructions, the adenovirus was modified so as to render it incapable of replication in the infected cell. Thus, the constructions described in the prior art are deleted adenoviruses from the E1 region, essential for viral replication, at the level of which heterologous DNA sequences are inserted (Levrero et al., Gene 101 (1991) 195; Gosh-Choudhury et al., Gene 50 (1986) 161). Furthermore, to improve the properties of the vector, it has been proposed to create other deletions or modifications in the genome of the adenovirus. Thus, a thermosensitive point mutation was introduced into the mutant ts125, making it possible to inactivate the 72kDa DNA binding protein (DBP) (Van der Vliet et al., 1975). Other vectors include a deletion of another region essential for viral replication and / or spread, the E4 region. The E4 region is in fact involved in the regulation of the expression of late genes, in the stability of late nuclear RNA, in the extinction of the expression of proteins of the host cell and in the efficiency of replication of l 'Viral DNA. Adenoviral vectors in which the E1 and E4 regions are deleted therefore have very reduced transcription background noise and expression of viral genes. Such vectors have been described by example in applications WO94 / 28152, WO95 / 02697, PCT / FR96 / 00088). In addition, vectors carrying a modification in the IVa2 gene have also been described (WO96 / 10088).

The recombinant adenoviruses described in the literature are produced from different adenovirus serotypes. There are in fact different serotypes of adenoviruses, the structure and properties of which vary somewhat, but which have a comparable genetic organization. More particularly, the recombinant adenoviruses can be of human or animal origin. As regards adenoviruses of human origin, mention may preferably be made of those classified in group C, in particular adenoviruses of type 2 (Ad2), 5 (Ad5), 7 (Ad7) or 12 (Adl2). Among the various adenoviruses of animal origin, mention may preferably be made of adenoviruses of canine origin, and in particular all the strains of adenoviruses CAV2 [manhattan or A26 / 61 strain (ATCC VR-800) for example]. Other adenoviruses of animal origin are cited in particular in application WO94 / 26914 incorporated herein by reference.

In a preferred embodiment of the invention, the recombinant adenovirus is a human adenovirus of group C. More preferably, it is an adenovirus Ad2 or Ad5. Recombinant adenoviruses are produced in an packaging line, that is to say a cell line capable of complementing in trans one or more of the deficient functions in the recombinant adenoviral genome. One of these lines is for example the line 293 in which a part of the adenovirus genome has been integrated. More specifically, line 293 is a human embryonic kidney cell line containing the left end (approximately 11-12%) of the genome of the adenovirus serotype 5 (Ad5), comprising the left ITR, the packaging region , the El region, including Ela and Elb, the region coding for the pIX protein and part of the region coding for the pIVa2 protein. This line is capable of transcomplementing recombinant adenoviruses defective for the E1 region, that is to say devoid of all or part of the E1 region, and to produce viral stocks having high titers. This line is also capable of producing, at permissive temperature (32 ° C.), stocks of virus further comprising the thermosensitive E2 mutation. Other cell lines capable of complementing the E1 region have been described, based in particular on human lung carcinoma cells A549 (WO94 / 28152) or on human retinoblasts (Hum. Gen. Ther. (1996) 215). Furthermore, lines capable of trans-complementing several functions of the adenovirus have also been described. In particular, mention may be made of lines complementing the El and E4 regions (Yeh et al., J. Virol. 70 (1996) 559; Cancer Gen. Ther. 2 (1995) 322; Krougliak et al., Hum. Gen. Ther. 6 (1995) 1575) and lines complementing the El and E2 regions (WO94 / 28152, WO95 / 02697, WO95 / 27071).

Recombinant adenoviruses are usually produced by introducing viral DNA into the packaging line, followed by lysis of the cells after approximately 2 or 3 days (the kinetics of the adenoviral cycle being 2Λ to 36 hours). After the cell lysis, the recombinant viral particles are isolated by centrifugation in a cesium chloride gradient.

For the implementation of the method, the viral DNA introduced may be the complete recombinant viral genome, optionally constructed in a bacterium (WO96 / 25506) or in a yeast (WO95 / 03400), transfected in the cells. It can also be a recombinant virus used to infect the packaging line. DNA viral can also be introduced in the form of fragments each carrying a part of the recombinant viral genome and a homology zone making it possible, after introduction into the packaging cell, to reconstitute the recombinant viral genome by homologous recombination between the different fragments. A conventional process for the production of adenovirus thus comprises the following steps: The cells (for example the 293 cells) are infected in a culture dish with a viral preset at the rate of 3 to 5 viral particles per cell (Multiplicity of Infection (MOI) = 3 to 5), or transfected with viral DNA. The incubation then lasts from 40 to 72 hours. The virus is then released from the nucleus by lysis of the cells, generally by several successive thawing cycles. The cell lysate obtained is then centrifuged at low speed (2000 to 4000 rpm) and the supernatant (clarified cell lysate) is then purified by centrifugation in the presence of cesium chloride in two stages:

- A first rapid centrifugation of 1.5 hours on two layers of cesium chloride of densities 1.25 and 1.40 framing the density of the virus (1.34) so as to separate the virus from the proteins of the medium;

- A second longer gradient centrifugation (from 10 to 40 hours depending on the rotor used), which constitutes the true and only step of purification of the virus.

Generally, after the second centrifugation step, the virus band is in the majority. However, two less dense, thin bands are observed, which observation by electron microscopy has shown that they were empty or broken viral particles for the densest band, and viral subunits (pentons, hexons) for the strip least den.se. After this step, the virus is harvested by needle puncture in the centrifuge tube and the cesium is removed by dialysis or desalting.

Although the levels of purity obtained are satisfactory, this type of process nevertheless has certain drawbacks. In particular, it is based on the use of cesium chloride, which is a reagent incompatible with therapeutic use in humans. Therefore, it is imperative to remove cesium chloride at the end of the purification. This process also has certain other drawbacks mentioned below, limiting its use on an industrial scale. To remedy these problems, it has been proposed to purify the virus obtained after lysis, not by cesium chloride gradient, but by chromatography. Thus, the article by Huyghe et al. (Hum. Gen. Ther. 6 (1996) 1403) describes a study of different types of chromatography applied to the purification of recombinant adenoviruses. This article describes in particular a study of purification of recombinant adenoviruses using low anion exchange chromatography (DEAE). Previous work has already described the use of this type of chromatography for this purpose (Klemperer et al., Virology 9 (1959) 536; Philipson L., Virology 10 (1960) 459; Haruna et al., Virology 13 (1961) 264). The results presented in the article by Huyghe et al. show a rather poor efficacy of the recommended ion exchange chromatography protocol. Thus, the resolution obtained is average, the authors indicating that virus particles are present in several chromatographic peaks; the yield is low (yield of viral particles: 67%; yield of infectious particles: 49%); and the viral preparation obtained following this chromatographic step is impure. In addition, pretreatment of the virus with different enzymes / proteins is necessary. This same article also describes a study of the use of gel permeation chromatography, demonstrating very poor resolution and very low yields (15-20%).

The present invention describes a new process for the production of recombinant adenoviruses. The process according to the invention results from modifications of the previous processes at the level of the production phase and / or at the level of the purification phase. The method according to the invention now makes it possible to obtain very quickly and industrially, stocks of viruses in very high quantity and quality.

One of the first aspects of the invention relates more particularly to a process for the preparation of recombinant adenoviruses in which the viruses are harvested from the culture supernatant. Another aspect of the invention relates to a process for the preparation of adenovirus comprising an ultrafiltration step. According to another aspect, the invention also relates to a method for purifying recombinant adenoviruses comprising a step of anion exchange chromatography. The present invention also describes an improved purification process using gas chromatography. gel permeation, optionally coupled to anion exchange chromatography. The method according to the invention makes it possible to obtain viruses of high quality, in terms of purity, stability, morphology and infectivity, with very high yields and under production conditions fully compatible with industrial requirements and with the regulations concerning the production of therapeutic molecules.

In particular, in terms of industrialization, the method of the invention uses methods for treating culture supernatants tested on a large scale for recombinant proteins, such as microfiltration or deep filtration, and tangential ultrafiltration. Furthermore, due to the stability of the virus at 37 ° C., this process allows better organization at the industrial stage since, unlike the intracellular method, the harvest time does not need to be precise at half day close. In addition, it guarantees a maximum harvest of the virus, which is particularly important in the case of defective viruses in several regions. On the other hand, the process of the invention allows easier and more precise monitoring of the production kinetics, directly on homogeneous samples of supernatant, without pretreatment, which allows better reproducibility of the productions. The method according to the invention also makes it possible to dispense with the step of lysis of the cells. Cell lysis has several drawbacks. Thus, it can be difficult to envisage breaking cells by freezing-thawing cycles on an industrial level. Furthermore, alternative lysis methods (Dounce, X-press, sonication, mechanical shearing, etc.) have drawbacks: they are potentially aerosol generators difficult to confine for L2 or L3 viruses (level of virus confinement, dependent their pathogenicity or their mode of dissemination), these viruses also tend to be infectious by air; they generate shear forces and / or release of heat which are difficult to control and which decrease the activity of the preparations. The solution of using detergents to lyse the cells would need to be validated and would also require validation of the detergent removal. Finally, cell lysis leads to the presence in the medium of numerous cellular debris, which makes the purification. In terms of virus quality, the method of the invention potentially allows better maturation of the virus leading to a more homogeneous population. In particular, since packaging of viral DNA is the last step in the viral cycle, premature lysis of cells potentially releases empty particles which, although not replicative, are a priori infectious and capable of p. participate in the toxic effect of the virus and increase the specific activity ratio of the preparations obtained. The specific infectivity ratio of a preparation is defined as the ratio of the total number of viral particles, measured by biochemical methods (DO260nm, HPLC, PCR, immunoenzymatic methods, etc.) over the number of viral particles generating an effect biological (formation of lysis plaques on cells in culture in solid medium, transduction of cells). In practice, for a purified preparation, this ratio is determined by making the ratio of the concentration of the particles measured by DO at 260 nm to the concentration of units forming the range of the preparation. This ratio must be less than 100.

The results obtained show that the process of the invention makes it possible to obtain a virus of a purity at least equal to its counterpart purified by centrifugation in cesium chloride gradient, in a single step and without prior treatment, starting from a concentrated viral supernatant.

A first object of the invention therefore relates to a process for the production of recombinant adenoviruses characterized in that the viral DNA is introduced into a culture of packaging cells and the viruses produced are harvested after release into the culture supernatant. Unlike the previous methods in which the viruses are harvested following a premature cell lysis carried out mechanically or chemically, in the method of the invention, the cells are not lysed by the intervention of an external factor. The culture is continued for a longer period of time, and the viruses are harvested directly from the supernatant, after spontaneous release by the packaging cells. The virus according to the invention is thus recovered in the cell supernatant, whereas in the previous methods, it is an intracellular virus, more particularly intranuclear. The applicant has now shown that, despite the lengthening of the duration of the culture and despite the use of larger volumes, the method according to the invention makes it possible to generate viral particles in high quantity and of better quality. In addition, as indicated above, this process makes it possible to avoid the lysis steps which are heavy at the industrial level and generate numerous impurities.

The principle of the process is therefore based on the collection of the viruses released in the supernatant. This process may involve a longer culture time than previous techniques based on cell lysis. As indicated above, the harvest time does not have to be precise to the nearest half day. It is essentially determined by the kinetics of release of the viruses into the culture supernatant.

The kinetics of virus release can be followed in different ways. In particular, it is possible to use analysis methods such as RP-HPLC, IE-HPLC, semi-quantitative PCR (example 4.3), staining of dead cells with trypan blue, measuring the release. of LDH-type intracellular enzymes, measurement of particles in the supernatant by Coulter-type devices or by light diffraction, immunological (ELISA, RIA, etc.) or nephelometric methods, titration by aggregation in the presence of antibodies , etc.

Preferably, the harvest is carried out when at least 50% of the viruses have been released into the supernatant. The time when 50% of the viruses have been released can be easily determined by carrying out kinetics according to the methods described above. Even more preferably, the harvest is carried out when at least 70% of the viruses have been released into the supernatant. It is particularly preferred to carry out the harvest when at least 90% of the viruses have been released into the supernatant, that is to say when the kinetics reach a plateau. The kinetics of virus release are essentially based on the replication cycle of the adenovirus, and can be influenced by certain factors. In particular, it can vary according to the type of virus used, and in particular according to the type of deletion carried out in the recombinant viral genome. In particular, the deletion of the E3 region appears to delay the release of the virus. Thus, in the presence of the E3 region, the virus can be harvested 24-48 hours after infection. In on the other hand, in the absence of the E3 region, a longer culture time seems necessary. In this regard, the applicant has carried out experiments on the kinetics of release of an adenovirus deficient for the E1 and E3 regions in the cell supernatant, and has shown that the release begins 4 to 5 days approximately post-infection, and lasts until on day 14 approximately. Release generally reaches a plateau between day 8 and day 14, and the titer remains stable for at least 20 days post-infection.

Preferably, in the method of the invention, the cells are cultured for a period of between 2 and 14 days. Furthermore, the release of the virus can be induced by expression in the packaging cell of a protein, for example viral, involved in the release of the virus. Thus, in the case of adenovirus, the release can be modulated by expression of the Death protein encoded by the E3 region of the adenovirus (protein E3-11.6K), optionally expressed under the control of an inducible promoter. Therefore, it is possible to reduce the time of release of the viruses and to collect in the culture supernatant, more than 50% of the viruses 24-48 hours post-infection.

To recover the viral particles, the culture supernatant is advantageously filtered beforehand. The adenovirus having a size of approximately 0.1 μm (120 nm), the filtration is carried out by means of membranes having a porosity sufficiently large to allow the virus to pass, but sufficiently fine to retain the contaminants. Preferably, the filtration is carried out by means of membranes having a porosity greater than 0.2 μm. According to a particularly advantageous embodiment, the filtration is carried out by successive filtrations on membranes of decreasing porosity. Particularly good results have been obtained by carrying out filtration on depth filters with decreasing porosity lOμm, l.Oμm then 0.8-0.2μm. According to another preferred variant, the filtration is carried out by tangential microfiltration on flat membranes or hollow fibers. More particularly, Millipore flat membranes or hollow fibers having a porosity of between 0.2 and 0.6 μm can be used. The results presented in the examples show that this filtration step has a yield of 100% (no loss of virus was observed by retention on the filter having the lowest porosity).

According to another aspect of the invention, the applicant has now developed a method for harvesting and purifying the virus from the supernatant. To this end, the supernatant thus filtered (or clarified) is subjected to ultrafiltration. This ultrafiltration makes it possible (i) to concentrate the supernatant, the volumes involved being significant, (ii) to carry out a first purification of the virus and (iii) to adjust the buffer of the preparation to the subsequent stages of preparation. According to a preferred embodiment, the supernatant is subjected to a tangential ultrafiltration. Tangential ultrafiltration consists in concentrating and fractionating a solution between two compartments, retentate and filtrate, separated by membranes of determined cutoff thresholds, by carrying out a flow in the retentate compartment of the device and by applying transmenbranary pressure between this compartment and the filtrate compartment. The flow is generally carried out by means of a pump in the retentate compartment of the device and the transbranar pressure is controlled by means of a valve on the liquid stream of the retentate circuit or of a variable flow pump on the liquid stream of the circuit filtrate. The speed of the flow and the transmembrane pressure are chosen so as to generate few shear forces (number of reynolds less than 5000 sec ^"1 , preferably less than 3000 sec ^{" 1} , pressure less than 1.0 bar) while avoiding clogging of the membranes. Different systems can be used to carry out ultrafiltration, such as, for example, spiral membranes (Millipore, Amicon), flat membranes or hollow fibers (Amicon, Millipore, Sartorius, Pall, GF, Sepracor). The adenovirus having a mass of approximately 1000 kDa, membranes having a cutoff threshold of less than 1000 kDa, preferably between 100 kDa and 1000 kDa, are advantageously used in the context of the invention. The use of membranes having a cutoff threshold of 1000 kDa or higher in fact leads to a significant loss of virus at this stage. Preferably, membranes with a cutoff threshold of between 200 and 600 kDa are used, even more preferably between 300 and 500 kDa. The experiments presented in the examples show that the use of a membrane having a cutoff threshold of 300 kDa allows the retention of more than 90% of viral particles while eliminating environmental contaminants (DNA, environmental proteins, cellular proteins, etc.). The use of a cutoff threshold of 500 kDa offers the same advantages.

The results presented in the examples show that this step makes it possible to concentrate large volumes of supernatant, without loss of virus (yield of 90%), and that it generates a better quality virus. In particular, concentration factors of 20 to 100 times can be easily obtained.

This ultrafiltration step thus constitutes an additional purification compared to the conventional scheme insofar as the contaminants of mass below the cut-off threshold (300 or 500 kDa) are eliminated at least in part. The improvement in the quality of the viral preparation is clear when the aspect of separation is compared after the first ultracentrifugation step according to the two methods. In the conventional method involving lysis, the tube of the viral preparation has a cloudy appearance with a coagulum (lipids, proteins) sometimes coming to touch the band of virus, while in the method of the invention, the preparation after release and ultrafiltration presents an already well resolved band of environmental contaminants which persist in the upper phase. The improvement in quality is also demonstrated when the profiles on ion exchange chromatography of a virus obtained by cell lysis are compared with the virus obtained by ultrafiltration as described in the present invention. Furthermore, it is possible to further improve the quality by continuing the ultrafiltration by a diafiltration of the concentrate. This diafiltration is carried out on the same principle as tangential ultrafiltration, and makes it possible to more completely remove contaminants of size greater than the cut-off threshold of the membrane, while achieving equilibration of the concentrate in the purification buffer.

Furthermore, the applicant has also shown that this ultrafiltration then makes it possible to purify the virus directly by chromatography on an ion exchange column or by gel permeation chromatography, making it possible to obtain an excellent resolution of the peak of viral particles without the need for treatment of the preparation prior to chromatography. This is particularly unexpected and advantageous. Indeed, as indicated in the article by Hyughe et al. abovementioned, the purification by chromatography of viral preparations gives poor results and further requires pretreatment of the viral suspension with benzonase and cyclodextrins.

More particularly, the method according to the invention is therefore characterized in that the viruses are harvested by ultrafiltration.

As indicated above, the resulting concentrate can be used directly for purifying the virus. This purification can be carried out by prior conventional techniques, such as centrifugation in a cesium chloride gradient or other ultracentrifugation medium making it possible to separate the particles according to their size, density or coefficient of sedimentation. The results presented in Example 4 indeed show that the virus thus obtained exhibits remarkable characteristics. In particular, according to the invention, it is possible to replace cesium chloride with a solution of iodixanol, 5.5 '- [(2-hydroxy-1-3propanediyl) -bis (acetylamino)] bis [N, N' -bis (2,3dihydroxypropyl-2,4,6-triodo-1,3-benzenecarboxamide] in which the virus sediments at equilibrium at a relative density between 1, 16 and 1.24. The use of this solution is advantageous because, unlike cesium chloride, it is not toxic, and the Applicant has also shown that, advantageously, the concentrate obtained also makes it possible to purify the virus directly by an ion exchange mechanism. or by gel permeation, and to obtain an excellent resolution of the chromatographic peak of viral particles, without the need for pretreatment.

According to a preferred embodiment, the viruses are therefore harvested and purified by anion exchange chromatography.

For anion exchange chromatography, different types of support can be used, such as cellulose, agarose (Sepharose gels), dextran (Sephadex gels), acrylamide (Sephacryl gels, Trisacryl gels), silica (TSK-gel SW gels), poly [styrene-divinylbenzene] (Source gels or Poros gels), the copolymer ethylene glycol-methacrylate (Toyopearl HW and TSK-gel PW gels), or mixtures (agarose-dextran: Superdex gel). Furthermore, to improve the chromatographic resolution, it is preferable within the framework of the invention to use supports in the form of beads, having the following characteristics: - as spherical as possible,

- of calibrated diameter (balls all identical or as homogeneous as possible), without imperfections or breaks,

- as small as possible in diameter: 10 μm beads have been described (MonoBeads from Pharmacia or TSK-gel from TosoHaas, for example). This value seems to constitute the lower limit for the diameter of the beads, the porosity of which must also be very high to allow the penetration of the objects to be chromatographed inside the beads (see below).

- while remaining rigid to resist pressure.

Furthermore, to chromatograph adenoviruses which constitute very large objects (diameter> 100 nm), it is important to use gels having an upper limit of high porosity, or even the highest possible, to allow access viral particles to the functional groups with which they are called upon to interact.

Advantageously, the support is chosen from agarose, dextran, acrylamide, silica, poly [styrene-divinylbenzene], the ethylene glycol-methacrylate copolymer, alone or as a mixture.

For anion exchange chromatography, the support used must be functionalized, by grafting a group capable of interacting with an anionic molecule. Most generally, the group consists of an ain which can be ternary or quaternary. By using a ternary amine, such as DEAE for example, a weak anion exchanger is obtained. By using a quaternary amine, a strong anion exchanger is obtained.

In the context of the present invention, it is particularly advantageous to use a strong anion exchanger. Thus, preferably according to the invention a chromatography support as used above is used, functionalized by quaternary amines. Among the supports operating with quaternary amines, examples that may be mentioned are Source Q, Mono Q, Q Sepharose, Poros HQ and Poros QE resins, Fractogel TMAE type resins, and Toyopearl Super Q resins. Preferred examples of resins usable in the context of the invention are the

Source, in particular Source Q, such as Q (Pharmacia), MonoBeads, such as Q (Pharmacia), resins of the Poros HQ and Poros QE type. The MonoBeads support (ball diameter 10 ± 0.5 μm) has been commercially available for more than 10 years and the Source type resins (15 μm) or Poros (10 μm or 20 μm) for approximately 5 years. These last two supports have the advantage of having a very wide distribution of internal pores (they range from 20 nm to 1 μm), thus allowing the passage of very large objects through the beads. In addition, they offer very little resistance to the circulation of liquid through the gel (therefore very little pressure) and are very rigid. The transport of solutes to the functional groups with which they will interact is therefore very rapid. The Applicant has shown that these parameters are particularly important in the case of the adenovirus, the diffusion of which is slow due to its size.

The results presented in the examples show that the adenovirus can be purified from the concentrate in a single step of anion exchange chromatography, that the purification yield is excellent (140% in terms of tdu, compared to the 49% value reported by Huyghes et al.) and that the resolution is excellent. In addition, the results presented show that the adenovirus obtained has a high infectivity, and therefore has the characteristics required for therapeutic use. Particularly advantageous results have been obtained with a strong anion exchanger, that is to say functionalized with quaternary amines, and in particular with Source Q resin. Source Q15 resin is particularly preferred. In this regard, another subject of the invention relates to a process for the purification of recombinant adenoviruses from a biological medium, characterized in that it comprises a purification step by strong anion exchange chromatography.

According to this variant, the biological medium can be a supernatant of packaging cells producing said virus, a lysate of packaging cells producing said virus, or a prepurified solution of said virus.

Preferably, the chromatography is carried out on a support functionalized with a quaternary amine. Still according to a preferred embodiment, the support is chosen from agarose, dextran, acrylamide, silica, poly [styrene-divinylbenzene], ethylene glycol-methacrylate copolymer, alone or as a mixture.

A particularly advantageous embodiment is characterized in that the chromatography is carried out on a Source Q resin, preferably Q15.

Furthermore, the method described above is advantageously carried out using a supernatant of producer cells, and comprises a prior step of ultrafiltration. This step is advantageously carried out under the conditions defined above, and in particular, it is a tangential ultrafiltration on a membrane having a cutoff threshold of between 300 and 500 kDa.

According to another embodiment of the method of the invention, the viruses are harvested and purified by gel permeation chromatography.

The gel permeation can be carried out directly on the supernatant, on the concentrate, or on the virus resulting from anion exchange chromatography. The supports mentioned for the anion exchange chromatography can be used in this step, but without functionalization.

In this regard, the preferred supports are agarose (Sepharose gels), dextran (Sephadex gels), acrylamide (Sephacryl gels, Trisacryl gels), silica (TSK-gel gels

SW), the ethylene glycol-methacrylate copolymer (Toyopearl HW and TSK-gel gels PW), or mixtures (agarose-dextran: Superdex gel). Particularly preferred supports are:

- Superdex 200HR (Pharmacia)

- Sephacryl S-500HR, S-1000HR or S-2000 (Pharmacia) - TSK G6000 PW (TosoHaas).

A preferred method according to the invention therefore comprises an ultrafiltration followed by anion exchange chromatography.

Another preferred method includes ultrafiltration followed by anion exchange chromatography, followed by gel permeation chromatography.

Another variant of the invention relates to a method for purifying adenovirus from a biological medium comprising a first stage of ultracentrifugation, a second stage of dilution or dialysis, and a third stage of anion exchange chromatography . Preferably, according to this variant, the first step is carried out by rapid ultracentrifugation on a cesium chloride gradient. The term rapid means ultracentrifugation ranging from about 0.5 to 4 hours. During the second stage, the virus is diluted or dialyzed against buffer, to facilitate its injection on the chromatography gel, and the elimination of the ultracentrifugation medium. The third step is carried out using anion exchange chromatography as described above, preferably strong anions. In a typical experiment, starting from the virus collected in the supernatant (or possibly intracellular), a 1st rapid ultracentrifugation is carried out with cesium chloride (as in Example 3). Then, after a simple dilution of the sample (for example by 10 volumes of buffer) or after a simple dialysis in buffer, the sample is chromatographed in ion exchange (as in example 5.1.). The advantage of this variant of the process of the invention comes from the fact that it implements 2 completely different modes of separation of the virus (density and surface charge), which can possibly bring the virus to a level of quality combining the performances of the 2 methods. In addition, the chromatography step allows simultaneously to eliminate the medium used for ultracentrifugation (cesium chloride for example, or any other equivalent medium mentioned above).

Another subject of the invention relates to the use of iodixanol, 5.5 ′ - [(2-hydroxy-1-3-propanediyl) -bis (acetylamino)] bis [N, N'-bis (2,3dihydroxypropyl-2, 4,6-triodo- 1, 3-benzenecarboxamide] for the purification of adenovirus.

For the implementation of the method of the invention, different adenovirus packaging cells can be used. In particular, the packaging cells can be prepared from different pharmaceutically usable cells, that is to say cultivable under industrially acceptable conditions and having no recognized pathogenic character. They can be established cell lines or primary cultures and in particular human retinoblasts, human lung carcinoma cells, or kidney embryonic cells. Advantageously, these are cells of human origin, which can be infected with an adenovirus. In this regard, mention may be made of KB, Hela, 293, Vero, gmDBP6, HER, A549, HER, etc. cells.

The cells of the KB line are derived from a human epidermal carcinoma. They are accessible to ATCC (ref. CCL17) as well as the conditions allowing their cultivation. The Hela human cell line comes from a carcinoma of the human epithelium. It is also accessible to ATCC (ref. CCL2) as well as the conditions allowing its cultivation. The cells of line 293 are human embryonic kidney cells (Graham et al., J. Gen. Virol. 36 (1977) 59). This line contains in particular, integrated into its genome, the left part of the genome of the human adenovirus Ad5 (12%). The gm DBP6 cell line (Brough et al., Virology 190 (1992) 624) consists of Hela cells carrying the E2 adenovirus gene under the control of the LTR of MMTV.

It can also be cells of canine origin (BHK, MDCK, etc.). In this regard, cells from the MDCK canine line are preferred. Culture conditions MDCK cells have been described in particular by Macatney et al., Science 44 (1988) 9.

Different packaging cell lines have been described in the literature and are mentioned in the examples. They are advantageously cells trans-complementing the El function of the adenovirus. Even more preferably, these are cells that complement the E1 and E4 or E1 and E2a functions of the adenovirus. These cells are preferably derived from human embryonic kidney or retinal cells, or from human lung carcinomas.

The invention thus provides a method of producing particularly advantageous recombinant adenoviruses. This method is suitable for the production of recombinant viruses defective for one or more regions, and in particular, viruses defective for the El region, or for the El and E4 regions. Furthermore, it is applicable to the production of adenoviruses of different serotypes, as indicated above.

According to a particularly advantageous mode, the method of the invention is used for the production of recombinant adenoviruses in which the E1 region is inactivated by deletion of a PvuII-BglII fragment going from nucleotide 454 to nucleotide 3328, on the sequence of l adenovirus Ad5. This sequence is accessible in the literature and also on the database (see in particular Genebank n ° M73260). In another preferred embodiment, the E1 region is inactivated by deletion of a HinfII-Sau3A fragment going from nucleotide 382 to nucleotide 3446. In a particular embodiment, the method allows the production of vectors comprising a deletion of the entire region E4. This can be achieved by excision of a Maell-Mscl fragment corresponding to nucleotides 35835-32720. In another particular mode, only a functional part of E4 is deleted. This part includes at least the ORF3 and ORF6 phases. By way of example, these coding phases can be deleted from the genome in the form of PvuII-AluI and BglII-PvuII fragments respectively, corresponding to nucleotides 34801-34329 and 34115-33126 respectively. The deletions of the E4 region of the Ad2 dl808 virus or of the Ad5 dll004, Ad5 dll007, Ad5 dllOll or Ad5 dll014 viruses can also be used in the context of the invention. In this regard, the cells of the invention are particularly advantageous for the production of viruses comprising an inactive E1 region and a deletion in the E4 region of the type of that present in the genome of Ad5 d11014, that is to say of E4 virus "retaining the ORF4 reading phase.

As indicated above, the deletion in the El region advantageously covers all or part of the El A and E1B regions. This deletion must be sufficient to render the virus incapable of autonomous replication in a cell. The part of the El region deleted in the adenoviruses according to the invention advantageously covers the nucleotides 454-3328 or 382-3446.

The positions given above refer to the sequence of the adenovirus

Wild Ad5 as published and accessible on the database. Although minor variations may exist between the different adenovirus serotypes, these positions are generally applicable to the construction of recombinant adenoviruses according to the invention from any serotype, and in particular adenoviruses Ad2 and Ad7.

Furthermore, the adenoviruses produced may have other alterations in their genome. In particular, other regions can be deleted to increase the capacity of the virus and reduce its side effects linked to the expression of viral genes. Thus, all or part of the E3 or IVa2 region in particular can be deleted. Regarding the E3 region, it may however be particularly advantageous to keep the part coding for the protein gpl9K. This protein in fact makes it possible to prevent the adenoviral vector from being the subject of an immune reaction which (i) limits its action and (ii) could have undesirable side effects. According to a particular mode, the E3 region is deleted and the sequence coding for the protein gpl9k is reintroduced under the control of a heterologous promoter.

As indicated before, adenoviruses constitute very efficient gene transfer vectors for gene and cell therapy applications. For this, a heterologous nucleic acid sequence whose transfer and / or expression in a cell, an organ or an organism is sought can be inserted into their genome. This sequence may contain one or more therapeutic genes, such as a gene whose transcription and possibly translation into the target cell generate products having a therapeutic effect. Among the therapeutic products, mention may more particularly be made of enzymes, blood derivatives, hormones, lymphokines: interleukins, interferons, TNF, etc. (FR 9203120), growth factors, neurotransmitters or their synthetic precursors or enzymes, trophic factors: BDNF, CNTF, NGF, IGF, GMF, aFGF, bFGF, NT3, NT5, etc; apolipoproteins: ApoAI, ApoAIV, ApoE, etc. (WO94 / 25073), dystrophin or a minidystrophin (WO93 / 06223), tumor suppressor genes: p53, Rb, RaplA, DCC, k-rev, etc. (WO94 / 24297 ), genes coding for factors involved in coagulation: Factors VII, VIII, IX, etc., suicide genes: thymidine kinase, cytosine deaminase, etc; or all or part of a natural or artificial immunoglobulin (Fab, ScFv, etc., WO94 / 29446), etc. The therapeutic gene can also be an antisense gene or sequence, the expression of which in the target cell makes it possible to control the expression of genes or the transcription of cellular mRNAs. Such sequences can for example be transcribed, in the target cell, into RNAs complementary to cellular mRNAs and thus block their translation into protein, according to the technique described in patent EP 140 308. The therapeutic gene can also be a gene encoding for an antigenic peptide capable of generating an immune response in humans, for the production of vaccines. These may in particular be antigenic peptides specific for the epstein barr virus, the HIV virus, the hepatitis B virus (EP 185 573), the pseudo-rabies virus, or even specific for tumors (EP 259 212).

Generally, the heterologous nucleic acid sequence also comprises a promoter region for functional transcription in the infected cell, as well as a region located 3 ′ of the gene of interest, and which specifies a transcriptional end signal and a site for polyadenylation. All of these elements constitute the expression cassette. As regards the promoter region, it may be a promoter region naturally responsible for the expression of the gene considered when it is capable of functioning in the infected cell. It can also be from regions of different origin (responsible for the expression of other proteins, or even synthetic). In particular, they may be promoter sequences of eukaryotic or viral genes or any promoter or derivative sequence, stimulating or repressing the transcription of a gene in a specific way or not and in an inducible way or not. By way of example, they may be promoter sequences originating from the genome of the cell which it is desired to infect, or from the genome of a virus, and in particular, the promoters of the E1A, MLP genes of adenovirus, the CMV promoter, LTR-RSV, etc. Among the eukaryotic promoters, mention may also be made of ubiquitous promoters (HPRT, vimentin, α-actin, tubulin, etc.), promoters of intermediate filaments (desmin, neurofilaments, keratin, GFAP, etc.) promoters of therapeutic genes (MDR type , CFTR, factor VIII, etc.) tissue-specific promoters (pyruvate kinase, villin, promoter of the intestinal fatty acid binding protein, promoter of actin to smooth muscle cells, specific promoters for the liver; Apo AI , Apo AU, Human albumin, etc.) or promoters responding to a stimulus (steroid hormone receptor, retinoic acid receptor, etc.). In addition, these expression sequences can be modified by adding activation, regulation sequences or allowing tissue-specific or majority expression. Furthermore, when the inserted nucleic acid does not contain expression sequences, it can be inserted into the genome of the defective virus downstream of such a sequence.

Furthermore, the heterologous nucleic acid sequence may also comprise, in particular upstream of the therapeutic gene, a signal sequence directing the therapeutic product synthesized in the secretory pathways of the target cell. This signal sequence may be the natural signal sequence of the therapeutic product, but it may also be any other functional signal sequence, or an artificial signal sequence.

The therapeutic gene expression cassette can be inserted at different sites in the genome of the recombinant adenovirus, according to the techniques described in the prior art. It can first of all be inserted at the level of the El deletion. It can also be inserted at the E3 region, in addition to or in substitution for sequences. It can also be located at the level of the deleted E4 region.

The present invention also relates to the purified viral preparations obtained according to the process of the invention, as well as any pharmaceutical composition comprising one or more defective recombinant adenoviruses prepared according to this process. The pharmaceutical compositions of the invention can be formulated for topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intraocular, transdermal, etc. administration.

Preferably, the pharmaceutical composition contains pharmaceutically acceptable vehicles for an injectable formulation. They may in particular be saline solutions (monosodium phosphate, disodium phosphate, sodium chloride, potassium, calcium or magnesium, etc., or mixtures of such salts), sterile, isotonic, or dry compositions, in particular lyophilized, which, by addition, as appropriate, of sterilized water or physiological saline, allow the constitution of injectable solutes. Other excipients can be used such as for example a hydrogel. This hydrogel can be prepared from any biocompatible and non-cytotoxic polymer (homo or hetero). Such polymers have for example been described in application WO93 / 08845. Some of them, such as in particular those obtained from ethylene oxide and / or propylene, are commercial. The doses of virus used for the injection can be adapted according to various parameters, and in particular according to the mode of administration used, the pathology concerned, the gene to be expressed, or even the duration of the treatment sought. In general, the recombinant adenoviruses according to the invention are formulated and administered in the form of doses between 10 ^ and 10 ^*** ^ pfu, and preferably 10 ° to 10 *** ^υ pfu. The term pfu ("plaque forming unit") corresponds to the infectious power of an adenovirus solution, and is determined by infection of an appropriate cell culture, and measures, generally after 15 days, the number of plaques of infected cells. The techniques for determining the pfu titer of a viral solution are well documented in the literature. Depending on the therapeutic gene, the viruses thus produced can be used for the treatment or prevention of many pathologies, including genetic diseases (dystrophy, cystic fibrosis, etc.), neurodegenerative diseases (Alzheimer, Parkinson, ALS, etc.), cancers , pathologies linked to coagulation disorders or dyslipoproteinemias, pathologies linked to viral infections (hepatitis, AIDS, etc.), etc.

The present invention will be more fully described with the aid of the examples which follow, which should be considered as illustrative and not limiting.

Legend of figures

Figure 1: Study of the stability of the purified adenovirus according to Example 4.

Figure 2: Analysis in HPLC (reverse phase) of the purified adenovirus according to Example 4. Comparison with the adenovirus of Example 3.

Figure 3: Release kinetics of the Ad-βGal adenovirus in the supernatant of 293 cells, measured by semi-quantitative PCR and Plate Assay. Figure 4: Elution profile on Source Q15 of an ultrafilter adenovirus supernatant (Example 5.1).

FIG. 5: Analysis in HPLC Resource Q of the peak of virus harvested by chromatography on resin Source Q15 of a supernatant of adenovirus ultrafilter (example 5.1). Figure 6: (A) Elution profile on Source Q15 resin of an adenovirus supernatant Ad-APOAl ultrafilter (Example 5.3); and (B) HPLC analysis (Resource Q) of the peak of harvested virus.

Figure 7: (A) Elution profile on Source Q15 of an adenovirus supernatant Ad-TK ultrafilter (Example 5.3). HPLC analysis (Resource Q) of the different fractions (beginning and end of peak) of harvested viruses: (B) F2 fraction, middle of the peak; (C) Fraction F3, peak bound; (D) Fraction F4, end of the peak.

Figure 8: Elution profile on Mono Q resin of a concentrated supernatant of culture of adenovirus producing cells (example 5.4). BG25F1: Supernatant virus concentrates and purifies on cesium. BG25C: Supernatant infects, concentrates. Figure 9 Elution profile on POROS HQ gel of a concentrated supernatant from the culture of adenovirus producing cells (Example 5 4) BG25F1 Supernatant virus concentrated and purified on cesium BG25C Infectant supernatant, concentrated

Figure 10 A and B Purification profile by gel permeation on Sephacryl S 1 OOOHR / Superdex 200HR of an adenovirus ultrafilter supernatant (Example 6)

Figure 1 1 Analysis by electron microscopy of a purified adenovirus stock according to the invention

Figure 12 Analysis by electron microscopy of the density 1 virus band 27

General molecular biology techniques

Classically used methods in molecular biology such as preparative extractions of plasmid DNA, centrifugation of plasmid DNA in cesium chloride gradient, electrophoresis on agarose or acrylamide gels, purification of DNA fragments by electroelution, the extraction of proteins with phenol or phenol-chloroform, the precipitation of DNA in a saline medium with ethanol or isopropanol, the transformation in Escherichia coli, etc. are well known to those skilled in the art and are abundantly described in the literature [Maniatis T et al, "Molecular Cloning, a Laboratory Manual", Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982, Ausubel FM et al (eds), "Current Protocols in Molecular Biology" , John Wiley & Sons, New York, 1987]

The plasmids of the pBR322, pUC type and the phages of the Ml 3 series are of commercial origin (Bethesda Research Laboratories) For the ligations, the DNA fragments can be separated according to their size by electrophoresis in agarose gels or acrylamide, extracts with phenol or with a phenol / chloroform mixture, precipitated with ethanol then incubated in the presence of phage T4 DNA ligase (Biolabs) according to the supplier's recommendations The filling of the prominent 5 'ends can be carried out by the Klenow fragment of E coli DNA Polymerase I (Biolabs) according to supplier's specifications Destruction of the prominent 3 'ends is carried out in the presence of phage DNA Polymerase T4 (Biolabs) used according to the manufacturer's recommendations. The destruction of the protruding 5 ′ ends is carried out by gentle treatment with nuclease SI.

Mutagenesis directed in vitro by synthetic oligodeoxynucleotides can be carried out according to the method developed by Taylor et al. [Nucleic Acids Res. 12 (1985) 8749-8764] using the kit distributed by Amersham. Enzymatic amplification of DNA fragments by the technique called PCR [Polymerase-catalyzed Chain Reaction, Saiki R.K. et al., Science 230 (1985) 1350-1354; Mullis K.B. and Faloona F.A., Meth. Enzym. 155 (1987) 335-350] can be performed using a "DNA thermal cycler" (Perkin Elmer Cetus) according to the manufacturer's specifications. Verification of the nucleotide sequences can be carried out by the method developed by Sanger et al. [Proc. Natl. Acad. Sci. USA, 74 (1977) 5463-5467] using the kit distributed by Amersham.

Examples

Example 1: Cell lines for packaging

The packaging cells used in the context of the invention can come from any cell line infectable with an adenovirus, compatible with use for therapeutic purposes. It is more preferably a cell chosen from the following lines:

- The cells of line 293: Line 293 is a line of human kidney embryonic cells containing the left end (approximately 11-12%) of the genome of the adenovirus serotype 5 (Ad5), comprising the left ITR, the packaging region, the El region, including Ela, Elb, the region coding for the pIX protein and part of the region coding for the pIVa2 protein (Graham et al., J. Gen. Virol. 36 (1977) 59 ). This line is capable of trans-complementing recombinant adenoviruses defective for the E1 region, that is to say devoid of all or part of the E1 region, and to produce viral stocks having high titers - The cells of the A549 line

Cells complementing the E1 region of the adenovirus were constructed from A549 cells (Imler et al., Gene Ther. (1996) 75). These cells contain a fragment of the El region, devoid of the left ITR, placed under the control of an inducible promoter.

- The cells of the HER line

Human retinal embryonic cells (HER) can be infected with an adenovirus (Byrd et al., Oncogene 2 (1988) 477). Adenovirus packaging cells prepared from these cells have been described for example in application WO94 / 28152 or in the article by Fallaux et al. (Hum.Gene Ther. (1996) 215). Mention may more particularly be made of the line 911 comprising the E1 region of the genome of the adenovirus Ad5, from nucleotide 79 to nucleotide 5789 integrated into the genome of HER cells. This cell line allows the production of viruses defective for the E1 region.

- IGRP2 cells

IGRP2 cells are cells obtained from 293 cells, by integration of a functional unit of the E4 region under the control of an inducible promoter. These cells allow the production of viruses defective for the E1 and E4 regions (Yeh et al., J. Virol (1996) 70).

- VK cells

VK cells (VK2-20 and VK10-9) are cells obtained from 293 cells, by integration of the entire E4 region under the control of an inducible promoter, and of the region coding for the protein pIX. These cells allow the production of viruses defective for the E1 and E4 regions (Krougliak et al., Hum. Gene Ther. 6 (1995) 1575).

- 293E4 cells

The 293E4 cells are cells obtained from 293 cells, by integration of the entire E4 region. These cells allow the production of viruses defective for the El and E4 regions (WO95 / 02697; Cancer Gene Ther. (1995) 322).

Example 2: Viruses used

The viruses produced in the context of the following examples are an adenovirus containing the LacZ marker gene from E. coli (Ad-βGal), an adenovirus containing the gene coding for the herpes virus type I thymidine kinase (Ad-TK ), an adenovirus containing the gene encoding the human p53 tumor suppressor protein and a virus encoding the apolipoprotein A1 (Ad-apoAI). These viruses are derived from the Ad5 serotype and have the following structure:

- A deletion in the E1 region covering, for example, nucleotides 382 (Hinfl site) to 3446 (Sau3a site).

- A gene expression cassette, under the control of the RSV or CMV promoter, inserted at the level of said deletion. - A deletion of the E3 region.

The construction of these viruses has been described in the literature (WO94 / 25073,

WO95 / 14102, FR 95.01632, Stratford-Perricaudet et al. J. Clin. Invest (1992) p626).

It is understood that any other construct can be produced according to the method of the invention, and in particular viruses carrying other heterologous genes and / or other deletions (E1 / E4 or E1 / E2 for example).

EXAMPLE 3 Production of Viruses by Lysis of Cells

This example reproduces the prior technique of virus production, consisting in lysing the packaging cells to recover the viruses produced.

The 293 cells are infected at 80-90% confluence in a culture dish with a preset of Ad-βGal or Ad-TK virus (example 2) at a rate of 3 to 5 viruses per cell (Multiplicity of Infection MOI = 3 to 5 ). The incubation lasts from 40 to 72 hours, the harvest timing being judged by the observation under the microscope of the cells which round, become more refractive and adhere more and more weakly to the culture support. In the literature, the kinetics of the viral cycle last from 24 to 36 hours.

At the level of a laboratory production it is important to harvest the cells before they detach in order to eliminate the medium of infection at the time of the harvest without losing cells and then to take them up in a minimum volume (the concentration factor is depending on the size of the culture of the order of 10 to 100 times).

The virus is then released from the nucleus by 3 to 6 successive thawing cycles (dry ice ethanol at -70 ° C, water bath at 37 ° C) The cell lysate obtained is then centrifuged at low speed (2000 at 4000 rpm) and the supernatant (clarified cell lysate) is then purified by ultracentrifugation in cesium chloride gradient in two stages:

- A first rapid ultracentrifugation (step) of 1.5 hours 35,000 rpm rotor sw 41, on two layers of cesium 1.25 and 1.40 framing the density of the virus (1.34) so as to separate the virus from the proteins in the medium; the rotors can be 'swinging' rotors (Sw28, S 41 Beckman) or fixed angle (Ti 45, Ti 70, Ti 70.1 Beckman) depending on the volumes to be treated;

- A second ultracentrifugation in a longer gradient (from 10 to 40 hours depending on the rotor used), for example 18 hours at 35,000 rpm in sw 41 rotor which constitutes the true and unique purification of the virus. The virus is found in a linear gradient at equilibrium at a density of 1.34.

Generally, at this stage, the virus band is in the majority. Sometimes, however, two less dense, thin bands are observed, which observation by electron microscopy has shown that they were empty or broken viruses and for the less dense band of viral subunits (pentons, hexons). After this stage, the virus is harvested in the tube by needle puncture and the cesium is eliminated by dialysis or desalting on G25.

EXAMPLE 4 Production of Viruses in the Supernatant This example describes an experiment in virus production by recovery after spontaneous release. The virus is then harvested by ultrafiltration, then purified by cesium chloride.

4.1. Protocol

In this method, unlike Example 3, the cells are not harvested 40 to 72 hours post-infection, but the incubation is prolonged between 8 to 12 days so as to obtain a total lysis of the cells without the need to proceed freezing and thawing cycles. The virus is found in the supernatant. The supernatant is then clarified by filtration through filters with decreasing porosity depth (10 μm / 1.0 μm / 0.8-0.2 μm).

The virus has a size of 0.1 μm and at this stage we did not observe any loss of virus by retention on the filter with the lowest porosity (0.22 μm)

The supernatant once clarified is then concentrated by tangential ultrafiltration on a Millipore spiral membrane having a cutoff threshold of 300 kDa. In the experiments reported in the present invention, the concentration factor is dictated by the dead volume of the system which is 100 ml. Volumes of supernatant from 4 to 20 liters were concentrated with this system, making it possible to obtain volumes of concentrate from 100 ml to 200 ml without difficulty, which corresponds to a concentration factor of 20 to 100 times. The concentrate is then filtered through 0.22 μm and then purified by centrifugation on cesium chloride as described in Example 3, followed by a dialysis step.

4.2. Results

Purity

While the intracellular virus tube (Example 3) presents a cloudy appearance with a coagulum (lipids, proteins) sometimes coming to touch the band of virus, the viral preparation obtained after the first stage of centrifugation on cesium chloride by the procedure of The invention presents a band of virus already well isolated from the environmental contaminants which persist in the upper phase. The analysis in high performance liquid chromatography on a Resource Q column (see example 5) also shows this gain in purity of the starting material obtained by ultrafiltration of infected supernatant with a reduction in nucleic acid contaminants (DO 260/280 ratio greater than or equal to 1.8) and protein (DO 260/280 ratio less than 1).

Stability of the virus in a supernatant at 37 ° C:

The stability of the virus was determined by titration by the plate assay method of an infectious culture supernatant from which aliquots were taken at different incubation times at 37 ° C. post-infection. The results are presented below:

Ad-TK virus:

. titer 10 days post-infection = 3.5 x 10 ⁸ pfu / ml

. titer 20 days post-infection = 3.3 x 10 ⁸ pfu / ml

Ad-βGal virus

. titer 8 days post-infection = 5.8 x 10 ⁸ pfu / ml

. titer 9 days post-infection = 3.6 x 10 ⁸ pfu / ml

. titrelO days post-infection = 3.5 x 10 ⁸ pfu / ml

. titer 13 days post-infection = 4.1 x 10 ⁸ pfu / ml

. titer 16 days post-infection = 5.5 x 10 ⁸ pfu / ml

The results obtained show that, up to at least 20 days post-infection, the titer of the supernatant is stable within the limit of precision of the assay. Furthermore, Figure 1 shows that, in the elution buffer, the virus is stable for at least 8 months, at -80 ° C. as at -20 ° C.

Specific infectivity of preparations This parameter, corresponding to the ratio of the number of viral particles measured by HPLC to the number of pfu, accounts for the infectious power of the viral preparations. According to FDA recommendations, it should be less than 100. This parameter was measured as follows: Two series of culture flasks containing 293 cells were infected in parallel at the same time with the same viral prestock under the same conditions. This experiment was carried out for a recombinant Ad-bgal adenovirus, then repeated for an Ad-TK adenovirus. For each adenovirus, a series of vials is harvested 48 hours post-infection and is considered to be a production of purified intracellular virus on a cesium gradient after freezing and thawing. The other series is incubated 10 days post-infection and the virus is harvested from the supernatant. The purified preparations obtained are titrated by assay plate and the quantification of the number of total viral particles is determined by measuring the concentration of PVII protein by reverse phase HPLC on a C4 Vydac 4.6 × 50 mm column after denaturation of the samples in 6.4M guanidine. . The quantity of PVII proteins is correlated with the number of viral particles considering 720 copies of PVII per virus (HorwitzNirology. Second edition (1990)). This method is correlated with the measurements of viral particles on preparations purified by the densitometric method at 260 nm taking as specific extinction coefficient 1.0 unit of absorbance = 1.1 10 ¹² particles per ml

The results obtained show that, for the Ad-βGal virus, this ratio is 16 for the supernatant virus and 45 for the intracellular virus. For the Ad-TK virus, the ratio is 78 in the case of the virus harvest in the supernatant method and 80 for the virus harvested in the intracellular method.

Analysis by electron microscopy:

This method makes it possible to detect the presence of empty particles or free co-purified viral units, as well as to assess a protein contamination of the purified viral preparations or the presence of non-dissociable aggregates of viral particles. Protocol: 20 μl of sample are placed on a carbon grid and then treated for observation in negative staining with 1.5% uranyl acetate. For observation, a Jeol 1010 electron microscope from 50 kV to 100 kV is used.

Result: The analysis carried out on a virus collected in the supernatant shows a clean preparation, without contaminants, without aggregates and without empty viral particles. It is also possible to distinguish the fibers of the virus as well as its regular geometric structure. These results confirm the high quality of the viral particles obtained according to the invention.

Protein profile analysis in HPLC and SDS PAGE:

Analysis in SDS PAGE:

20μl of sample is diluted in Laemmli buffer (Nature 227 (1970) 680-685), reduced 5 min at 95 ° c, then loaded onto Novex gels 1 mm x 10 wells 4-20% gradient. After migration, the gels are stained with coomassie blue and analyzed on Image Master VDS Pharmacia. The analysis reveals an electrophoretic profile for the virus collected in the supernatant in accordance with the data from the literature (Lennart Philipson, 1984 H.S Ginsberg editors, Plenum press, 310-338).

Analysis in reverse phase HPLC:

Figure 2 shows the overlay of 3 chromatograms obtained from two virus samples collected intracellularly and a virus sample purified by the supernatant method. The experimental conditions are as follows: Vydac column ref 254 Tp 5405, RPC44.6x50 mm, SolvantXA: H2O + TFA0.07%; Solvent B: CH3CN + TFA 0.07%, linear gradient: T = 0min% B = 25; T = 50min% B = 50%; Flow = lml / min, detector = 215 nm. The chromatograms show a perfect identity between the samples, with no difference in the relative intensities of each peak. The nature of each peak was determined by sequencing and demonstrates that the proteins present are all of viral origin (see table below). PIC (Min) IDENTIFICATION

19-20 PVII precursor

21-22 Precursor PVII; PX precursor 1 to 12

27-28 PVI precursor; PX precursor

32-33 PX Precursor

34

35-36 mature PVII

37 mature PVII; PVIII precursor

39-41 mature PVI

45 pX

46 pIX

In vitro analysis of transduction efficiency and cytotoxicity

The cytotoxicity analysis is carried out by infecting HCT116 cells in 24-well plates for increasing MOIs and by determining the percentage of living cells compared to an uninfected control, 2 and 5 days post-infection, using the technique of staining with violet crystal. The results are shown in the table below:

Adenovirus ME = 3.0 ME = 10.0 ME = 30.0 ME = 100.0

Supernatant J2 91% 96% 87% 89%

SumageantJ5 97% 90% 10% <5%

Transduction efficiency analysis

For an AD-βGal adenovirus, the transduction efficiency of a preparation is determined by infecting W162 cells, non-permissive to replication, cultured in 24-well plates, with increasing concentrations of viral particles. For the same quantity of viral particles deposited, there are, 48 hours post-infection, the cells expressing the betagalactosidase activity after incubation with X-gal as substrate. Each blue cell is counted as a transduction unit (TDU), the result is multiplied by the dilution of the sample in order to obtain the concentration in unit of transductions of the sample. The transduction efficiency is then expressed by making the ratio of the concentration of viral particles to the concentration of TDU. The results obtained show that the purified viruses exhibit good transduction efficiency in vitro.

In vivo intracerebral expression analysis

In order to evaluate the efficiency of the adenoviruses according to the invention for the transfer and expression of genes in vivo, the adenoviruses were injected stereotaxically into the striatum of OF1 immunocompetent mice. For this, volumes of lμl to 10 'pfu of virus were injected at the following stereotaxic landmarks (for the 0mm incisor bar): Anteroposterior: +0.5; Medio-lateral: 2; Depth: -3.5.

The brains were analyzed 7 days after the injection. The results obtained show that the transduction efficiency is high: thousands of transduced cells, very intense expression in the nucleus and frequent and intense diffusion in the cytoplasm.

4.3. Kinetics of virus release

This example describes a study of the kinetics of release of adenoviruses in the culture supernatant of packaging cells.

This study was carried out by semi-quantitative PCR using oligonucleotides complementary to regions of the adenovirus genome. For this purpose, the linearized viral DNA (1-10 ng) was incubated in the presence of dXTP (2 μl, 10 mM), of a pair of specific oligonucleotides and of Taq Polymerase (Cetus) in a 10XPCR buffer, and subjected to 30 amplification cycles without the following conditions: 2 min at 91C, 30 cycles (1 min 91C, 2 min at annealing temperature, 3 min at 72C), 5 min 72C, then 4C. PCR experiments were carried out with the pairs of oligonucleotides of sequence: - Couple 1:

TAATTACCTGGGCGGCGAGCACGAT (6368) - SEQ ID n ° 1

ACCTTGGATGGGACCGCTGGGAACA (6369) - SEQ ID n ° 2

- Couple 2:

TTTTTGATGCGTTTCTTACCTCTGG (6362) - SEQ ID n ° 3

CAGACAGCGATGCGGAAGAGAGTGA (6363) - SEQ ID n ° 4

- Couple 3:

TGTTCCCAGCGGTCCCATCCAAGGT (6364) - SEQ ID n ° 5

AAGGACAAGCAGCCGAAGTAGAAGA (6365) - SEQ ID n ° 6

- Couple 4:

GGATGATATGGTTGGACGCTGGAAG (6366) - SEQ ID n ° 7

AGGGCGGATGCGACGACACTGACTT (6367) - SEQ ID n ° 8

The amount of adenovirus released into the supernatant was determined on a supernatant of 293 cells infected with Ad-βGal, at different post-infection times. The results obtained are shown in Figure 3. They show that cell release begins from the 5th or 6th day post infection.

It is understood that any other virus determination technique can be used for the same purpose, on any other packaging line, and for any type of adenovirus.

EXAMPLE 5 Purification of the Virus by Ultrafiltration and Ion Exchange This example illustrates how the adenovirus contained in the concentrate can be purified directly and in a single chromatographic step of ion exchange, with very high yields.

5.1. Protocol

In this experiment, the starting material therefore consists of the concentrate

(or ultrafiltration retentate) described in Example 4. This retentate has a total protein content of between 5 and 50 mg / ml, and more preferably between 10 and 30 mg / ml, in PBS buffer (10 mM phosphate , pH 7.2 containing 150 mM NaCl).

The ultrafiltration supernatant obtained from a virus preparation is injected into a column containing Source Q 15 (Pharmacia) equilibrated in 50 mM Tris / HCl buffer pH 8.0 containing 250 mM NaCl, 1.0 mM MgC12, and 10% glycerol (buffer A). After rinsing with 10 column volumes of buffer A, the adsorbed species are eluted with a linear gradient of NaCl (250 mM to 1 M) on 25 column volumes at a linear flow rate of 60 to 300 cm / h, more preferably 12 cm / h. The typical elution profile obtained at 260 nm is presented in FIG. 4. The fraction containing the viral particles is collected. It corresponds to a fine, symmetrical peak, the retention time of which coincides with the retention time obtained with a preparation of viral particles purified by ultracentrifugation. It is possible to inject, under the conditions described above, at least 30 mg of total proteins per ml of Source Q 15 resin while retaining an excellent resolution of the peak of viral particles.

In a representative experiment carried out using a preparation of an β-gal adenovirus (Example 2), 12.6 mg of total protein was injected into a Resource Q column (1 ml), ie 5 × 10 ′ PFU and 1.6 x 10 ¹⁰ TDU. The peak of viral particles collected after chromatography (3.2 ml; Fig. 5) contained 173 μg of protein and 3.2 x 10 ¹⁰ PFU and 2.3 x 10 '° TDU. The viral particles have therefore been purified 70 times (in terms of quantity of proteins) and the purification yield is 64% in PFU and 142% in TDU (See Table below).

Steps Concentration Volumes Volumes Returns Sample factor deposits collected Purification

SURNANTANT 5000 ml 5000 ml 100%

ULTRAProteins: 6.3 mg / ml 5000 ml 200 ml 100% 5

PFU FILTRATION: 2.5xl0 ¹⁰ / ml

300 kd TDU: 8.1x 10 ⁹ / ml Particle 3.8 xlθ "/ rnl

Share / pfu ratio: 16.0 Share / t ratio: 47.0

PFU: 1.0x 10 ¹⁰ / ml Proteins = 85%

PURIFICATION TDU: 7.2xl0 ⁹ / ml 2.0 ml of 3.0 ml PFU = 64% 70

EXCHANGE Particle: 2.0xl0 ^{! i} / ml elution concentrate TDU = 140%

IONS Particles = 84%

(one step) Part / pfu ratio = 20 Part / tdu ratio = 27 HPLC Purete = 98.4%

PFU: 1.0xl0 ⁿ / ml) 28.3 ml of QSP 4.1 PFU = 66%

GRADIENT TDU: 7.5x 10 ^IO / ml concentrate ml TDU = 130% 70

CsCl Particle: 2.2xl0 ¹² / ml Particles = 84%

Share / pfu ratio = 22 HPLC per / t of ratio = 29 purity = 98.4%

5.2. Purity

After this purification step, the fraction collected has a purity of 98% in viral particles (UV detection at 260 nm), when it is analyzed by high performance liquid chromatography (HPLC) on a column of Resouce Q (1 ml) in a following chromatographic system: 10 μl of the fraction purified by chromatography as described in Example 5.1 are injected onto a Resource Q15 column (1 ml of gel; Pharmacia) balanced in 50 mM Tris / HCl buffer pH 8.0 (buffer B) . After rinsing with 5 ml of buffer B, the adsorbed species are eluted with a linear gradient of 30 ml of NaCl (0 to 1 M) in buffer B at a flow rate of 1 ml / min. The eluted species are detected at 260 nm. This HPLC analysis (Figure 5) further shows that the residual bovine serum albumin present in the ultrafiltration retentate is completely eliminated during the preparative chromatography. Its content in the purified fraction is estimated to be <0.1%. Western blot analysis with a polyclonal anti-BSA antibody (with ECL disclosure; Amersham) indicates that the BSA content in the chromatographic preparation is less than 100 ng per mg of virus.

The electrophoresis analysis of the purified adenoviral fraction by chromatography is carried out on polyacrylamide gel (4-20%) under denaturing conditions (SDS). The protein bands are then revealed with silver nitrate.

This analysis shows that the adenoviral preparation obtained by chromatography has a level of purity at least equal to that of the preparation conventionally maintained by ultracentrifugation since it does not have an additional protein band which would indicate contamination of the preparation with non-adenoviral proteins.

The adenoviral preparation obtained by chromatography has an absorbance ratio A ₂ 60 nm At 280 nm equal ^to 1.30 ± 0.05. This value, which is identical to that obtained for the best preparations obtained by ultracentrifugation, indicates that the preparation is devoid of contaminating proteins or contaminating nucleic acids.

The analysis by electron microscopy carried out under the conditions described in Example 4.2 on an Ad-βgal virus purified by chromatography shows a clean preparation, without contaminants, without aggregates and without empty viral particles (FIG. 11). In addition, the ultracentrifugation in cesium chloride gradient of this preparation reveals a single band of density 1.30, which confirms the absence of contamination of the chromatographic preparations by possible empty particles or fragments of capsids. During purification, the chromatographic peak of the virus is followed by a shoulder (or secondary peak) in its rear part, which is not collected with the main peak. The cesium chloride gradient ultracentrifugation of this fraction reveals a density band 1.27, and analysis of the composition of this fraction shows that it does not contain nucleic acids. Analysis by electron microscopy shows that this fraction contains irregularly shaped particles, with perforations on the surface (Figure 12). They are therefore empty particles (devoid of DNA) and incomplete. This therefore demonstrates that the purification of the adenovirus by chromatography eliminates the empty particles present in small quantities in the preparations before purification.

5.3. Purification of adenoviruses comprising a therapeutic gene such as the genes coding for the ApoAl proteins or thimidine kinase.

This example illustrates how adenoviruses comprising in their genomes heterologous nucleic acid sequences encoding proteins can be purified directly and in a single chromatographic ion exchange step. It also shows that the chromatographic behavior of the adenovirus is independent of the heterologous nucleic acid sequences that it carries, which makes it possible to implement the same purification process for different adenoviruses carrying diverse heterologous nucleic acid sequences.

In a typical purification experiment, an adenovirus comprising in its genome a heterologous nucleic acid sequence coding for the ApoAl protein (Example 2, WO94 / 25073) is purified by chromatography, in the system described in Example 5.1, 18 ml (72 mg of proteins; 1.08 x 10 ³ particles) of concentrated supernatant from a cell culture harvested 10 days post-infection (FIG. 6A). The peak of viral particles collected after chromatography (14 ml; 1.4 mg of protein) contained 9.98 x 10 ¹² particles, which indicates a particle yield of 92% and a purification factor of 51. After this step of purification, the fraction collected exhibited (Figure 6B) a purity greater than 98% in viral particles during the chromatographic analysis under the conditions described in 5.2. Analysis by electrophoresis of the adenoviral fraction purified by chromatography carried out under the conditions described in Example 5.2. showed that this preparation had a level of purity at least equal to that of the preparation conventionally obtained by ultracentrifugation, and that it is devoid of contaminating proteins or contaminating nucleic acids.

In another typical purification experiment, an adenovirus comprising in its genome a heterologous nucleic acid sequence coding for the herpes simplex protein thimidine kinase type 1 (Example 2, WO95 / 14102) is purified by chromatography in the system described in Example 5.1. 36 ml (180 mg protein; 4.69 x 10 ¹³ particles) of concentrated cell culture supernatant (Figure 7A). The peak of viral particles collected after chromatography (20 ml; 5.6 mg of protein) contained 4.28 x 10 ⁿ particles, which indicates a particle yield of 91% and a purification factor of 32. After this step of purification, the collected fraction presented (Figures 7B-D) a purity greater than 99% in viral particles during the chromatographic analysis under the conditions described in Example 5.2. and an absorbance ratio of 1.29. The electrophoresis analysis of the purified adenoviral fraction by chromatography carried out under the conditions described in Example 5.2. showed that this preparation had a level of purity at least equal to that of the preparation conventionally obtained by ultracentrifugation, and that it is devoid of contaminating proteins or contaminating nucleic acids.

5.4. Purification of intracellular adenovirus by strong anion exchange.

This example illustrates how an adenovirus comprising in its genome a heterologous nucleic acid sequence can be purified directly in a single chromatographic step of ion exchange from a lysate of packaging cells producing said virus.

In a typical purification experiment, an adenovirus comprising in its genome a heterologous nucleic acid sequence coding for the β-gal protein is purified by chromatography in the system described in example 5.1 (column

FineLine Pilot 35, Pharmacia, 100 ml of Source 15Q resin), 450 ml (i.e. 2.5 x 10 * ⁴ particles) of concentrated lysate from a cell culture harvested 3 days post-infection by chemical lysis (1% Tween-20). The peak of viral particles collected after chromatography (110 ml) contained 2.15 x 10 ⁴ particles, which indicates a particle yield of 86%. After this purification step, the fraction collected had a purity greater than 98% in viral particles during the chromatographic analysis under the conditions described in 5.2. The electrophoresis analysis of the purified adenoviral fraction by chromatography carried out under the conditions described in Example 5.2 showed that this preparation had a level of purity at least equal to that of a preparation obtained conventionally by ultracentrifugation, and that it is devoid of contaminating proteins or contaminating nucleic acids. 5.5. Purification of the virus by ultrafiltration and ion exchange chromatography on different columns.

This example illustrates how the adenovirus contained in the concentrate can be purified directly and in a single chromatographic ion exchange step using a gel different from the Source 15 Q support, while operating on the same separation principle, anion exchange by interaction with quaternary amino groups in the matrix.

In a typical adenovirus purification experiment, different recombinant adenoviruses (coding for βGal, apolipoprotein AI and TK) were purified by chromatography on a column of Source Q30 gel using the protocol described in Example 5.1 . The results obtained show that the Source Q30 gel makes it possible to obtain viral preparations with a purity of the order of 85%, with a yield of between 70 and 100%. In addition, the results obtained show that Q30 has, for the purification of adenoviruses, an efficiency (expressed by the number of theoretical platings) of 1000 and a (maximum quantity of virus which can be chromatographed without the peaks being altered. ) from 0.5 to 1 x 10 ¹² pv per ml. These results show that the Source Q30 gel can therefore be suitable for the purification of recombinant adenoviruses, even if its properties remain inferior to those of Source Q15 (purity on the order of 99%, efficiency on the order of 8000 and ability to 2.5 to 5 10 ¹² pv per ml).

In another typical adenovirus purification experiment, a β-Gal adenovirus is purified by chromatography on a column of MonoQ HR 5/5 according to the protocol described in Example 5.1. The chromatographic image corresponding to the ultrafiltration retentate and to the purified viral preparation thus obtained is illustrated in FIG. 8.

In another typical adenovirus purification experiment, a β-Gal adenovirus is purified by chromatography on a Poros HQ / M column following the protocol described in Example 5.1. The corresponding chromatographic image to the ultrafiltration retentate and to the purified viral preparation thus obtained is illustrated in FIG. 9.

EXAMPLE 6 Purification of the Virus by Ultrafiltration and Gel Permeation

This example illustrates how the adenovirus contained in the concentrate (ultrafiltration retentate) can be purified directly by gel permeation chromatography, with very high yields.

6.1. Protocol

200 μl of the ultrafiltration retentate obtained in example 4 (ie 1.3 mg of proteins) are injected into an HR 10/30 column (Pharmacia) filled with Sephacryl S-1000SF (Pharmacia) balanced for example in PBS buffer , pH 7.2 containing 150 mM NaCl (buffer C). The species are fractionated and eluted with buffer C at a flow rate of 0.5 ml / min and detected at the outlet of the column in UV at 260 nm. Alternatively, under the same conditions of use, a column filled with Sephacryl S-2000 can be used, which allows better resolution than the Sephacryl S-1000HR column for particles from 100 nm to 1000 nm.

The resolution of the two gel permeation chromatographic systems described above can be advantageously improved by chromatographing the ultrafiltration supernatant (200 μl) on a system of 2 columns HR 10/30 (Pharmacia) coupled in series (Sephacryl S column -1000HR or S-2000 followed by a column of Superdex 200 HR) equilibrated in buffer C. The species are eluted with buffer C at a flow rate of 0.5 ml / min and detected in UV at 260 nm. In this system, the peak of viral particles is very clearly better separated from the lower molecular weight species than in the system comprising a Sephacryl S-1000 HR or Sephacryl S-2000 column alone. In a representative experiment, an ultrafiltration retentate (200 μl, 1.3 mg of protein) was chromatographed on a system of 2 columns Sephacryl S-ÎOOOHR-Superdex 200 HR 10/30 (Figure 10). The chromatographic peak containing the viral particles was collected. Its retention time coincides with the time of retention obtained with a preparation of viral particles purified by ultracentrifugation. The peak of viral particles collected after chromatography (7 ml) contained 28 μg of protein and 3.5 10 ⁹ PFU. Its analysis by analytical ion exchange chromatography under the conditions described in Example 5.2. shows the presence of a contaminating peak more strongly retained on the analysis column, the surface of which represents approximately 25% of the surface of the viral peak. Its 260nm / 280nm absorbance ratio which has a value of 1.86 indicates that this contaminating peak corresponds to nucleic acids. The viral particles were therefore purified approximately 50 times (in terms of quantity of proteins) and the purification yield is 85% in PFU.

Alternatively, it is possible to chromatograph the preparations of viral particles (ultrafiltrates or fractions at the output of anion exchange chromatography) on a TSK G6000 PW column (7.5 x 300 mm; TosoHaas) balanced in buffer C. species are eluted with buffer C at a flow rate of 0.5 ml / min and detected in UV at 260 nm. Similarly, it may be advantageous to increase the resolution of the chromatographic system, in particular to increase the separation of the peak of viral particles from the lower molecular weight species, by chromatographing the ultrafiltration supernatant (50 to 200 μl) on a system. of 2 columns coupled in series [column of TSK G6000 PW (7.5 x 300 mm) followed by a column of Superdex 200 HR] balanced in buffer C. The species are eluted with buffer C at a flow rate of 0, 5 ml / min and detected in UV at 260 nm.

Example 7: Purification of the virus by Ultrafiltration. Ion exchange and gel permeation

The fraction of viral particles resulting from anion exchange chromatography (example 5) can advantageously be chromatographed in one of the gel permeation chromatographic systems described above, for example in order to further improve the level purity of the viral particles, but also mainly for the purpose of conditioning the viral particles in a compatible or adapted medium for subsequent uses of the viral preparation (injection, etc.). LIST OF SEQUENCES

(1) GENERAL INFORMATION:

(l) DEPOSIT:

(A) NAME: Rhône Poulenc Rorer SA

(B) STREET: 20, avenue Raymond Aron

(C) CITY: ANTONY (E) COUNTRY: France

(F) POSTAL CODE: 920165

(n) TITLE OF THE INVENTION: PROCESS FOR PRODUCING RECOMBINANT ADENOVIRUSES.

(m) NUMBER OF SEQUENCES: 8

(iv) COMPUTER-DETACHABLE FORM:

(A) TYPE OF MEDIA: Floppy disk (B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS / MS-DOS

(D) SOFTWARE: Patentin Release # 1.0, Version # 1.30 (EPO)

(2) INFORMATION FOR SEQ ID NO: 1:

(î) CHARACTERISTICS OF THE SEQUENCE: (Λ) LENGTH: 25 base pairs

(B) TYPE: nucleotide (C) NUMBER OF STRANDS: Single

(D) CONFIGURATION: linear

(il) TYPE OF MOLECULE: DNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1:

TAATTACCTG GGCGGCGAGC ACGAT 25

(2) INFORMATION FOR SEQ ID NO: 2:

(î) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleotide

(C) NUMBER OF STRANDS: simple (D) CONFIGURATION: linear

(il) TYPE OF MOLECULE: DNA

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2:

ACCTTGGΛTG GGACCGCTGG GAACA 25

(2) INFORMATION FOR SEQ ID NO: 3: (î) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleotide

(C) NUMBER OF STRANDS: single

(D) CONFIGURATION: linear

(il) TYPE OF MOLECULE: DNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3: TTTTTGATGC GTTTCTTACC TCTGG 25

(2) INFORMATION FOR SEQ ID NO: 4:

(î) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 25 base pairs (B) TYPE: nucleotide

(C) NUMBER OF STRANDS: single

(D) CONFIGURATION: linear

(il) TYPE OF MOLECULE: DNA

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4:

CAGACAGCGΛ TGCGGAAGAG AGTGA 25

(2) INFORMATION FOR SEQ ID NO: 5:

(1) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleotide (C) NUMBER OF STRANDS: single

(D) CONFIGURATION: linear

(il) TYPE OF MOLECULE: DNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 5:

TGTTCCCAGC GGTCCCATCC AAGGT 25

(2) INFORMATION FOR SEQ ID NO: 6:

(î) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleotide

(C) NUMBER OF STRANDS: simple (D) CONFIGURATION: linear

(n) TYPE OF MOLECULE: DNA

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 6:

AAGGACAAGC AGCCGAAGTA GAAGA 25

(2) INFORMATION FOR SEQ ID NO: 7: (1) CHARACTERISTICS OF THE SEQUENCE: (Λ) LENGTH: 25 base pairs

(B) TYPE: nucleotide

(C) NUMBER OF STRANDS: single

(D) CONFIGURATION: linear

(il) TYPE OF MOLECULE: DNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 7: GGATGATATG GTTGGACGCT GGAAG 25

(2) INFORMATION FOR SEQ ID NO: 8:

(î) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleotide (C) NUMBER OF STRANDS: single

(D) CONFIGURATION: linear

(n) TYPE OF MOLECULE: DNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 8:

AGGGCGGΛTG CGΛCGACACT GACTT 25

Claims

1. Method for producing recombinant adenoviruses characterized in that the viral DNA is introduced into a culture of packaging cells and the viruses produced are harvested after release into the supernatant.

2. Method according to claim 1 characterized in that the harvest is carried out when at least 50% of the viruses have been released in the supernatant.

3. Method according to claim 1 characterized in that the harvest is carried out when at least 70% of the viruses have been released in the supernatant.

4. Method according to claim 1 characterized in that the harvest is carried out when at least 90% of the viruses have been released in the supernatant.

5. Method according to claim 1 characterized in that the viruses are harvested by ultrafiltration.

6. Method according to claim 5 characterized in that the ultrafiltration is a tangential ultrafiltration.

7. Method according to claim 5 or 6 characterized in that the ultrafiltration is carried out on a membrane having a cutoff threshold less than 1000 kDa.

8. Method according to claim 1 characterized in that the viruses are harvested by anion exchange chromatography.

9. Method according to claim 8 characterized in that the anion exchange chromatography is a strong anion exchange chromatography.

10. Method according to claim 9 characterized in that the strong anion exchange chromatography is carried out on a support chosen from Source Q, Mono Q, Q Sepharose resins, Poros HQ and Poros QE, Fractogel TMAE type resins and Toyopearl Super Q.

11. Method according to claim 1 characterized in that the viruses are harvested by gel permeation chromatography.

12. Method according to claim 11 characterized in that the gel permeation chromatography is carried out on a support chosen from gels Sephacryl S-500 HR, Sephacryl S- 1000 SF, Sephacryl S- 1000 HR, Sephacryl S-2000, Superdex 200 HR, Sepharose 2B, 4B or 6B and TSK G6000 PW.

13. Method according to claim 1 characterized in that the viruses are harvested by ultrafiltration followed by anion exchange chromatography.

14. The method of claim 13 characterized in that the viruses are harvested by ultrafiltration followed by anion exchange chromatography and then by gel permeation chromatography.

15. Method according to one of the preceding claims, characterized in that the packaging cell is a cell trans-complementing the El function of the adenovirus.

16. The method of claim 15 characterized in that the packaging cell is a cell trans-complementing the El and E4 functions of the adenovirus.

17. The method of claim 15 characterized in that the packaging cell is a cell trans-complementing the El and E2a functions of the adenovirus.

18. Method according to one of claims 15 to 17 characterized in that the cell is an embryonic human kidney cell, a human retinoblast or a cell of a human carcinoma.

19. Method for purifying recombinant adenoviruses from a biological medium, characterized in that it comprises a purification step by strong anion exchange chromatography.

20. The method of claim 19 characterized in that the biological medium is a supernatant of packaging cells producing said virus.

21. The method of claim 19 characterized in that the biological medium is a lysate of packaging cells producing said virus.

22. The method of claim 19 characterized in that the biological medium is a prepurative solution iee of said virus.

23. The method of claim 19 characterized in that the chromatography is carried out on a support functionalized with a quaternary amine.

24. The method of claim 23 characterized in that the support is chosen from agarose, dextran, acrylamide, silica, polyfstyrene-divinylbenzene], ethylene glycol-methacrylate copolymer, alone or as a mixture.

25. The method of claim 24 characterized in that the chromatography is performed on a Source Q, Mono Q, Q Sepharose, Poros HQ, Poros QE, Fractogel TMAE, or Toyopearl Super Q resin.

26. The method of claim 25 characterized in that the chromatography is carried out on a Source Q resin, preferably Source Q15.

27. The method of claim 19 characterized in that it comprises a prior ultrafiltration step.

28. The method of claim 27 characterized in that the ultrafiltration is a tangential ultrafiltration on the membrane having a cutoff threshold between

300 and 500 kDa.

29. Purified viral preparation obtained according to the method of claim 1 or 19.

30. A pharmaceutical composition comprising a viral preparation according to claim 29 and a pharmaceutically acceptable vehicle.

31. Use of iodixanol, 5.5 '- [(2-hydroxy-1-3-propanediyl) -bis (acetylamino)] bis [N, N' -bis (2,3dihydroxypropyl-2,4,6-triodo-1, 3-benzenecarboxa mi de] for the purification of adenovirus.

32. Process for the purification of adenovirus from a biological medium comprising a first stage of ultracentrifugation, a second stage of dilution or dialysis, and a third stage of anion exchange chromatography.