WO2002061103A1 - Method for obtaining packaging cells producing retrovirus by eliminating cells liable to be infected by the retrovirus produced - Google Patents

Abstract

The invention concerns a method for obtaining retrovirus packaging cells non-permissive to infection by said retrovirus or a retrovirus of the same type. The invention also concerns the use of a retroviral vector for implementing said method and a method for preparing non-replicative retrovirus by transfecting the packaging cells obtained by said method.

Description

PROCESS FOR OBTAINING RETROVIRUS-PRODUCING ENCAPSIDATION CELLS BY ELIMINATING INFECTIBLE CELLS BY THE RETROVIRUS PRODUCED

The present invention relates to the field of biology, and more particularly to the field of virology. The invention relates to a method for obtaining retrovirus packaging cells which are transcomplementing and not permissive to infection by said retrovirus. The invention also relates to the use of retroviral vector for the implementation of said method of obtaining as well as a method of

Preparation of a non-replicative recombinant retrovirus by transfection of packaging cells obtained by said production process.

The use of non-replicating retroviral vectors is growing more and more in basic research

15 only in the clinic. The production of non-replicating vectors requires the use of cell lines called "transcomplementing". These lines were developed to produce all of the structural proteins necessary to assemble a retrovirus but do not produce

They do not spontaneously virus themselves. The development of these lines is generally long and tedious and involves many steps in molecular biology and cell culture. When we introduce, by transfection into these lines, a genome

25 viral whose structural genes have been replaced by genes of interest, one obtains the production of viral particles which carry the genes in question. These retroviral vectors can cycle through retroviral infection but are not able to emerge from the infected cell in the form of a viral particle because the genes for viral structure are absent.

One of the most common problems encountered with the use of these lines is the progressive loss of the ability to produce the virus and the difficulty in obtaining cell clones producing large amounts of virus. This "drift" occurs in naive transcomplementing lines as in producer cells. This phenomenon, well known to users, requires more or less regular subcloning of the cells and testing them to isolate clones having more favorable characteristics. These constraints encountered in all types of use take time and are an obstacle to the use of vectors. To avoid this, it is generally recommended to use viral stocks from early passages. However, it is sometimes difficult for laboratories to have access to such cell lines having a low number of culture passages. Some authors have imagined producing these vectors transiently. This technique, especially used in gene therapy, consists in cotransfecting at the same time a plasmid carrying the structural genes and another plasmid containing the vector which one wishes to produce. The vectors are harvested over a period of 48 to 72 hours after which the culture is eliminated. Viral stocks are then produced which are frozen before use. However, this type of production can only be implemented for certain viral species which support freezing well and for which highly transfectable cell lines have been isolated which cannot encapsulate all types of vectors.

To overcome the drawbacks of using retroviral species in culture, the inventors have developed a process based on the phenomenon of "interference retroviral ”to quickly and surely eliminate parasitic cells from cell culture, the presence of which contributes to greatly reducing the efficiency of viral production. The term “viral interference phenomenon” is intended to denote the resistance of the packaging lines to infection by the vectors which they produce, since the retroviral envelope proteins produced by the cells block the cellular receptor necessary for the entry of the virus in the cell. The inventors have postulated that the cells which derive lose the expression of the genes of viral structure.

The inventors have developed a system based on retroviral interference in which cells which express little or no viral structure genes can be eliminated from the culture. The basic principle is to use a retroviral vector to infect the cells of the packaging line. The cells which will have been infected will be those which will have lost the expression of the genes of viral structure. They can therefore be eliminated from the culture by various means which will be listed below.

The present invention therefore proposes to provide a process for obtaining retrovirus packaging cells, trans-complementary non-permissive to infection by said retrovirus, characterized in that it comprises the steps of:

(i) culturing a population of said packaging cells;

(ii) introduction by retroviral infection into the said cells cultivated in (i) of a retroviral vector comprising at least one “marker” gene included in the encapsidable retroviral genome;

(iii) selection of said cells which do not express said “marker” gene. For the purposes of the present invention, the term “retroviruses” is intended to denote oncogenic and non-oncogenic viruses naturally present in nature which affect animals and humans, variants of its retroviruses originating from instability and from the intrinsic genetic variety or from rearrangement between retroviruses, as well as recombinant retroviruses obtained by genetic engineering.

The fundamental characteristics of retroviruses are to have genetic information stored in the form of RNA and to have at the 3 'and 5' ends two untranslated sequences oriented in the same way (LTR, for "Long Terminal Repeat"), as well as to use reverse transcriptase during their multiplication. The genetic RNA of a retrovirus contains sequences of genes acting by trans coding for viral proteins: (i) a structural protein GAG which associates with RNA in the body of the viral particle; (ii) POL reverse transcriptase which catalyzes the synthesis of complementary DNA ( _C DNA); and (iii) an ENV envelope glycoprotein which is located in the bilayer of the particles and which binds the virus to the surface of host cells during infection. Replication of the retrovirus is regulated by cis-acting elements, such as the promoter for transcription of proviral DNA, and other nucleotide sequences necessary for viral replication. The elements acting in cis are present in or next to the LTRs. Retroviruses replicate by copying their RNA genome by reverse-transcription into double-stranded intermediate DNA using reverse-transcriptase. Intermediate DNA is integrated into the chromosomal DNA of the host cell. Integrated retroviral DNA is called a "provirus". The provirus serves as a matrix for the synthesis of RNA chains for the formation of infectious viral particles. The retroviruses according to the invention are used as retroviral vectors by replacing, in the proviral form of the retrovirus, one or more endogenous genes acting in trans with a sequence or a recombinant gene of interest. Genes acting in trans include the GAG, POL and ENV genes necessary for the production of intact virions. Recombinant retroviruses, deficient in GAG, POL and ENV “transactivator” genes, cannot therefore synthesize essential proteins for their replication and are therefore defective for replication. The term “defective virus” is intended to denote a virus which has lost any of the functions which it requires to ensure its perpetuation, either in its proviral form or in a vegetative form. Such recombinant retroviruses defective for replication are propagated using packaging cell lines or by cotransfection with “helper” viruses.

The term “retroviral vector or recombinant proviral DNA sequence within the meaning of the present invention” is intended to denote a retroviral nucleotide sequence having conserved the essential CIS sequences, that is to say the LTRs for the control of transcription and integration, the sequence or sequences necessary for encapsidation, the PBS sequence (“Primer binding site”) necessary for viral replication, and which is deleted from at least one viral gene GAG, POL, ENV, optionally replaced by at least a DNA sequence of interest.

A taxonomy of retroviruses has been described by Coffin JM, Hughes SH, armus HE (EDS.), Retroviruses, Cold Spring Harbor Laboratory Press (1997), appendix 2, pages 757-775. A non-exhaustive list of retroviruses according to the invention is given below: Avian Leukosis Virus (ALV), Rous Sarcoma Virus (RSV), Rous-Associated Virus (RAV), Fujinami Sarcoma Virus (FuSV), Avian Myelocytoma Avian Erythroblastosis Virus (AEV), S13 virus (MH2)

Erythroblastosis Virus (AEV), Avian Myeloblastosis Virus

(AMV), Avian Retrovirus RPL12, CT10 Avian Sarcoma Virus

(CT10), Avian Myeloblastosis-Erythoblastosis Virus E26 (E26), Avian Myelocytoma MC29 (MC29), Avian Retrovirus

RPL30, Rous-Associated Virus-0 (RAV-0), Avian Sarcoma Virus

(UR2), Avian Retrovirus (SKV), Y73 Avian Sarcoma Virus

(Y73), Avian Sarcoma Virus 17, Avian Retrovirus AS42

(ASV42), Avian Retrovirus (ASV31), Mouse Mammary Tumor Virus (MMTV), Gross Murine Leukemia Virus (Gross MLV), Graffi Murine Leukemia Virus (GraffiMLV), Friend Murine Leukemia Virus (FR-MLV), Radia tion Leukemia Virus ( RadLV), Spleen Necrosis Virus (SNV), Moloney Murine Leukemia Virus (Mo-MLV), Harvey Murine Sarcoma Virus (Ha-MSV), Feline Leukemia Virus (Fe-LV), Spleen Focus-Forming Virus (SFFV), Finkel - Biskis-Jinkins Murine Sarcoma Virus (FB-MSV), Moloney Murine Sarcoma Virus (Mo-MSV), Avian

Reticuloendotheliosis Virus (REV), Kirsten Murine Sarcoma Virus (Ki-MSV), Baboon Endogenous Virus (BaEV), Abelson Murine Leukemia Virus (Ab-MLV), Gibbon Ape Leukemia Virus

(GALV), Gardner-Arnstein Féline Sarcoma Virus (GA-FeSV),

McDonough Feline Sarcoma Virus (SM-FeSV), Simian Sarcoma

Virus (SSV), Snyder-Theilen Féline Sarcoma Virus (ST-FeSV),

Murine Sarcoma virus 3611 (MSV3611), Hardy-Zuckerman Feline Sarcoma Virus (HZ4FeSV), Mouse Myeloproliferative Leukemia Virus, Mason-Pfizer Monkey-Type Virus (MPMV), Jaagsiekte virus, Bovine Leukemia Virus (BLV), Human T-Cell Leukemia Virus -1 (HTLV-1), Human T-Cell Leukemia Virus-2 (HTLV-2), Equine Infectious Anemia Virus (EIAV), Visnavirus, Caprine Arthrisis-Encephalitis Virus (CAEV), Bovine

Immunodeficiency Virus (BIV), Human Immunodeficiency Virus- 1 (HIV-1), Simian Immunodeficiency Virus (SIV), Human Immunodeficiency Virus-2 (HIV-2), Feline Immunodeficiency Virus (FIV), Human Foamy Virus (HFV), Simian Foamy Virus (SFV), Walleye Dermal Sarcoma virus (WDSV).

According to a preferred embodiment, the retrovirus according to the invention is the spleen necrosis virus (SNV). The proviral DNA and the retroviral vector according to the invention also derives from the SNV. The SNV genome is for example available in the form of a plasmid (PpblO1) under the number ATCC 45012.

The retroviruses, retroviral vector, proviral DNA according to the invention, are either amphotropic, to allow their use in different species or cell types, or ecotropic.

It should be emphasized that the present invention is more particularly intended for the use of retroviral vectors and the corresponding packaging cells. However, it is also within the scope of the invention to provide a method according to the invention suitable for other types of viral vectors, such as for example adenoviral or adenoviral-associated vectors, used in fundamental research, in genetic engineering or in gene therapy , and which use surface proteins or receptors to enter the host cell.

By packaging line, within the meaning of the present invention, is meant a cell line which contains all or part of one or more retroviral genomes integrated or not in the cell genome and which supply the gene sequences acting in trans necessary for the production intact virions. Thus, a recombinant proviral DNA sequence containing a DNA sequence of interest, introduced into an encapsidation cell line by transfection or infection are then transcribed into RNA and encapsulated into infectious virions containing the sequence of interest; such infectious virions being unable to synthesize the proteins GAG, POL, ENV to carry out the packaging of viral RNA in particles capable of infecting other cells, the resulting infectious retroviral vectors can therefore infect other cells and integrate the recombinant proviral DNA sequence of interest into the cellular DNA of a host cell, but they cannot replicate. The term “non-permissive” means a cell which does not allow the virus to enter said cell, thus not allowing the virus to multiply and reproduce new viruses. The non-permissiveness of the cells according to the invention and a non-permissiveness of infection. The term “trans-complementant” is intended to denote the packaging cells which express in a transient or permanent, inducible or constitutive manner, at least one retroviral transactivating gene GAG, POL, ENV whose expression product is necessary for the assembly of the retrovirus in the cell.

The term “transfection” is intended to denote any experimental technique consisting in causing a nucleotide sequence to penetrate into a eukaryotic cell where the transfection techniques are known to those skilled in the art. Mention may be made, in a non-exhaustive manner, of calcium phosphate precipitation, electroporation, lipofection, thermal shock, the use of cationic polymers such as DEAE-dextran, polybrene, polyethylene glycol. By infection is meant the transfer of genetic material from one cell to another via a virus. According to a preferred embodiment, said retroviral vector is introduced into packaging cells by retroviral infection. The packaging line according to the present invention can be derived from cells of any animal or plant species. Preferably, these cells are derived from murine cells, monkey cells, avian cells, human cells. By way of example, a non-exhaustive list of vector packaging cells retrovirals is given below; this list is not exhaustive and should in no way limit the present invention. Among the murine packaging cells, mention should be made of cells derived from (i) NIH 3T3 cells, and their derived cells such as, for example, ATCC CRL 9078, ATCC CRL 10686 cells, (ii) COS cells and their derived cells. , (iii) 293 cells and their derived cells such as ATCC CRL 11270, ATCC CRL 11268, ATCC CRL 11269, ATCC CRL 11554. Among human packaging cells, mention should be made of human fibrosarcoma cells, in particular HT cells. 1080 and their derived cells. The packaging cells according to the present invention have receptors on their cell surface which participate in the entry of retroviruses into the cell. Preferably, these receptors bind a wide spectrum of viruses, thus allowing the packaging cells according to the invention to be infected with a broad spectrum of viruses, in particular of retroviruses. Conversely, it may be advantageous to have packaging cells whose cellular receptors for the virus are specific for a single viral species.

According to a first embodiment of the invention, the method according to the invention is characterized in that said selection is carried out by eliminating, from said population of packaging cells, the cells expressing said "marker" gene. According to this first embodiment, said “marker” gene is a “suicide” gene whose cellular expression causes the death of the cells expressing said “suicide” gene. By suicide gene according to the present invention is meant a nucleic acid coding for a protein product, characterized in that this product causes cell death by itself, or in the presence of another compound, such as for example a prodrug. This is the reason why, the method according to the invention is characterized in that it also comprises the step of administering a prodrug in the culture medium in a sufficient amount so that said prodrug after being metabolized by the expression product of said “suicide” gene is converted into a compound toxic to said cell expressing said gene “ marker pen ". The “suicide” gene according to the present invention corresponds inter alia to the genes used by a person skilled in the art in molecular biology to carry out a negative cell selection (for review, see US Pat. No. 5,627,059). Said "suicide" gene according to the invention preferably codes for a protein chosen from the group consisting of kinases, bacterial cytosine deaminase, hypoxanthine phosphoribosyl transferase (HPRT) and guanine phosphoribosyl transferase (GpT) ). More particularly, said kinase is chosen from the group consisting of thymidine kinase from the Herpes simplex virus and thymidine kinase from the varicella virus (“Varicella zoster virus”). Preferably, it is the thymidine kinase of the herpes simplex virus type I; when the kinase used is the herpes simplex virus type I thymidine kinase, the pro-drug is chosen from the group consisting of acyclovir, ganciclovir, FIAU (1- (2-deoxy-2-fluoro) - β-D-arabinofuranosyl) 5-iodouracil or their derivatives When the kinase used is the thymidine kinase of the chickenpox virus, the pro-drug is chosen from the group consisting of 6-methoxypurine arabinoside or one of When the protein expressed from said suicide gene is the bacterial cytosine deaminase, the prodrug is chosen from the group composed of 5-fluorocytosine and its derivatives which are converted into 5-fluorouracil, a compound highly toxic for When the protein expressed from said suicide gene is HPRT (Hypoxanthine phosphoribosyl transferase, the drug is 6-thio-guanine and its derivatives. When the protein expressed from said suicide gene is guanine phosphoribosyl transferase (GpT), the pro-drug is 6-thio-xanthine and its derivatives. When the suicide gene according to the present invention codes for a protein product which by itself causes cell death, the said protein product can be a toxin, such as the diphtheria toxin or the castor toxin.

According to a second embodiment of the invention, the method according to the invention is characterized in that said “marker” gene is a “killer” gene which codes for a pro-apoptotic protein whose cellular expression causes the death of cells expressing said “marker” gene. The term “pro-apoptotic proteins” is intended to denote the proteins which intervene in apoptosis or promote apoptosis. Among the pro-apoptotic proteins, mention should be made of the proteins of the Bcl2 family, and more particularly the BIK proteins

("Bcl2-Interacting Protein"), BAX (Oltvai et al. 1993), BAK (Chittenden et al. 1995; Kiefer et al. 1995) and BID ("BH3-Interacting Domain Death Agonist") (Wang et al. 1996 ). Among the pro-apoptotic proteins, mention should also be made of caspases, the protein AIF (“Apoptosis-Inducing Factor”) (Susin et al. 1999) and proteins of the tumor necrotizing factor family (TNF, “Tumor Necrosing Factor ”), and more particularly TNF itself (Old 1985), the protein FASL (“ FAS-Ligand ”) (Takahashi et al. 1994).

According to a third embodiment of the invention, the method according to the invention is characterized in that said selection is carried out by sorting from said population of packaging cells the cells which express and which do not express said gene "marker ". According to this third embodiment, the method is characterized in that said marker gene is a “reporter” gene whose cellular expression makes it possible to sort the cells expressing said selection gene. The term “reporter” gene is intended to denote a gene which allows the cells comprising this gene to be detected specifically, that is to say to be distinguished from other cells which do not carry this marker gene. Said “reporter” gene according to the invention codes for a protein preferably chosen from the group composed of auto-fluorescent proteins, such as the green fluorescence protein (GFP, for “Green Fluorescence Protein”), the red fluorescence protein ( RFP for "Red Fluorescence Protein"), as well as the variants of these fluorescence proteins obtained by mutagenesis to generate a fluorescence of different color. Said "reporter" gene also codes for any enzyme detectable fluorescently, phosphorescently, or visible by a histochemical process on living cells. Non-exhaustively, mention should be made of β-galactosidase (β-GAL), β-glucoronidase (β-GUS), alkaline phosphatase, in particular placental alkaline phosphatase (PLAP), alcoholic dehydrogenase, in particular alcoholic dehydrogenase of Drosophila (ADH), luciferase, especially

Firefly Luciferase, chloramphenicol-acetyl transferase (CAT), growth hormone (GH). When said marker gene is a “reporter” gene, it is advantageous to sort the population of packaging cells, instead of eliminating certain cells from the culture. This is the reason why, the present invention also relates to the method according to the invention characterized in that it further comprises the step of sorting the population of packaging cells. By way of nonlimiting example, this sorting can be carried out by means of a cell sorter.

(FACS for "Fluorescence Analysis Cell Sorter"). But other devices or technologies intended for carrying out cell sorting, known to a person skilled in the art, can be used.

According to another embodiment of the invention, the method according to the invention is characterized in that said retroviral vector further comprises a second selection gene. The term “selection gene” is intended to denote a gene which allows the cells which possess it to be specifically selected for or against the presence of a corresponding selective agent. To illustrate this, an antibiotic resistance gene can be used as a positive selection marker gene which allows a host cell to be positively selected in the presence of the corresponding antibiotic. A variety of positive and negative markers are known to those skilled in the art (for review see US Patent 5,627,059). This selection gene can be found either inside or outside the packable retroviral genome. When the second selection gene is located inside the encapsidable retroviral genome, that is to say between the sequences LTR5 'and LTR3', the latter may be present in the form of a gene entity distinct from the gene marker pen. In this case, said second selection gene is operably linked with DNA sequences making it possible to control the expression of said second gene. These sequences, known to those skilled in the art, correspond in particular to promoter sequences, optionally to activator sequences, to transcription termination signals to splicing sequences. Optionally, said second selection gene can constitute a gene for fusion with the marker gene. Said fusion gene is then operably linked with DNA sequences making it possible to control the expression of said fusion gene. According to another embodiment of the invention, said second gene for selection of said retroviral vector is located outside the encapsidable retroviral genome. As described in the examples below, the localization of said selection gene outside the encapsidable retroviral genome makes it possible to increase the maximum level of clones producing retroviral particles independently of the rate of drift of the packaging line. Said second selection gene according to the invention is preferably chosen from the antibiotic resistance genes. Among the antibiotics, neomycin, tetracycline, ampicillin, kanamycin, phleomycin, bleomycin, hygromycin, chloramphenicol, carbenicillin, geneticin should be mentioned in a non-exhaustive manner. The resistance genes corresponding to these antibiotics are known to those skilled in the art; for example, the bacterial aminoglycoside phosphotransferase (APH) gene makes cells resistant to the presence of the antibiotic neomycin in the culture medium. The second selection gene can also be selected from the HisD gene, the corresponding selective agent being histidinol. The second selection gene can also be selected from the guanine phosphoribosyl transferase (GpT) gene, the corresponding selective agent being xanthine. The second selection gene can also be selected from the hypoxanthine phosphoribosyl transferase (HPRT) gene, the corresponding selective agent being hypoxanthine. According to another embodiment of the invention, the second selection gene is selected from the group of genes coding for metabolic enzymes for which there are mutant packaging cells which lack them, for example the gene for dihydrofolate reductase for dhfr cells, _^"the. thymidine kinase gene for TK ^~ cells. the transformants are then selected for their ability to grow in selective medium such as hypoxanthine-aminopterin medium thymidine (HAT) for example. Thus, among the genes coding for metabolic enzymes, it is worth mentioning in a non-exhaustive manner thymidine kinase, dihydrofolate reductase, hygromycin β-phospho-transferase, xanthine-guanine phosphoribosyl transferase

(XGPRT), CAD (carbamyl phosphate synthase, aspartate transcarbamylase, dihydroorotase), adenosine deaminase, asparagine synthetase.

It may also be advantageous for the retroviral vector according to the invention to further comprise at least one third gene located inside the encapsidable retroviral genome. Said third may be present in the form of a gene entity distinct from the marker gene or constitute a gene for fusion with said marker gene. Said third gene encoding an enzyme may be particularly useful in estimating the titer of the retroviral preparation. This is why the method according to the invention is characterized in that said retroviral vector further comprises a gene coding for an enzyme located inside the encapsidable retroviral genome. According to this particular embodiment of the invention, the method further comprises a step of titrating the viral particles by assaying the activity of said enzyme. Enzyme activity assay techniques are widely known to those skilled in the art of biochemistry. These techniques preferably use photometric, fluorimetric, isotopic, immunological, radioimmunological, manometric assays (for review, see P. Kamoun "Apparatus and Methods in Biochemistry", 3rd Ed. Medicine-Sciences Flammarion). The enzymes capable of being used in the process according to the invention are known to a person skilled in the art; they are preferably enzymes which catalyze the transformation of a product into a substrate whose quantitative and qualitative determination is easy and quick. Among the enzymes according to the invention, non-exhaustive mention should be made of alcohol dehydrogenase, and in particular alcohol dehydrogenase from Drosophila, lacticodehydrogenase, transaminases, such as glutamate-pyruvate transaminase.

The invention also relates to the use of retroviral vector for the implementation of a process for obtaining cells for the packaging of non-permissive trans-complementary retroviruses, characterized in that said vector comprises at least one marker gene such as as previously defined and chosen from suicide genes, killer genes, “reporter” genes located between the sequences LTR5 'and LTR3'. Optionally, but preferably, said vector comprises a second selection gene and / or a third gene.

The invention also relates to a process for preparing a non-replicating recombinant retrovirus, characterized in that said process comprises the following steps:

(i) obtaining trans-complementary non-permissive packaging cells by implementing a method according to the invention;

(ii) transfection into said cells obtained in (i) of a retroviral vector comprising at least the encapsidable genome of said recombinant retrovirus;

(iii) culturing the cells thus transfected under conditions allowing the expression of the packaging proteins; (iv) harvesting of said recombinant retroviruses.

According to another embodiment of the invention, it is possible to introduce the encapsidable genome of the recombinant retrovirus into the encapsidation cells by viral infection. Said retroviral vector used in the above preparation process comprises at least one sequence or a gene of interest. Optionally, it comprises at least one positive or negative selection gene; preferably said selection gene is located outside the encapsidable retroviral genome. More preferably, it is a positive selection gene for antibiotic resistance; in this case said culture of step iii) is carried out in the presence of said antibiotic. The invention also relates to the non-replicating recombinant retrovirus obtained by the above preparation process. The retrovirus is prepared in packaging cells selected by the production process according to the invention which uses retroviruses of the same viral species. However, the use of amphotropic lines allows the use of different viral species.

In general, in the retroviral vectors of the present invention, the marker gene, and / or the selection gene, and / or said third gene and / or the gene sequence of interest are operably linked to elements controlling their expression. By elements controlling expression is meant to denote all the DNA sequences involved in the regulation of gene expression, that is to say the minimal promoter sequence, the upstream sequences, the activator sequences

("Enhancers"), possibly the inhibitory sequences

(“Silencers”), “insulator” sequences, splicing sequences. The elements controlling expression allow either a constitutive, ubiquitous, inducible expression, specific for a cell type ("tissue-specific") or specific for a stage of development.

The article by Metzger and Feil (1999) gives by way of nonlimiting example (cf. table on page 471) a list of tissue-specific cellular promoters susceptible to be used to direct the expression of gene sequences in different tissues. The tissue-specific expression elements are preferably chosen from promoters which make it possible to obtain a specific, and preferably strong, expression in one or more cell (s), tissue (s), cell type (s) ) or organ (s) of the organism according to the invention.

The ubiquitous expression elements or ubiquitous promoters are chosen from promoters which make it possible to obtain expression, preferably strong, in the whole, or at least in a large proportion of organs, or of tissues of the organism. according to the invention. By way of nonlimiting example of ubiquitous promoters, mention may be made of the cytomegalovirus (CMV) promoter (Schmidt et al., 1990) and the interferon-inducible promoter (Mxl) (Hug et al., 1998) early and immediate promoter of the human cytomegalovirus (HCMV-IE) (Boshart et al., 1987), the beta-actin promoter (Gunning et al. 1984), the histone H4 promoter (Guild et al. 1988), the murine metallothionein promoter (Me Ivor et al., 1987), rat growth hormone promoter (Miller et al., 1985), human adenosine deaminase promoter (Hantzapoulos et al., 1989) , the HSV thymidine kinase promoter (Tabin et al., 1982), the activator (“enhancer”) of the alpha-1 antitrypsin gene (Peng et al., 1988), the immunoglobulin promoter / enhancer (Blankenstein et al. 1988), the early or late promoters of SV40, the major late promoter of adenovirus 2 or other viral promoters derived from polyoma virus, bovine papilloma virus or other retroviruses or other adenoviruses. In addition, among the expression elements, or promoters according to the invention, some can provide constitutive or inducible control of the expression of the gene of interest. From elements ensuring an inducible expression, mention should be made of eukaryotic promoters inducible by heavy metals (Mayo et al., 1982; Brinster et al., 1982; Seark et al., 1985), by thermal shock (Nover et al. , 1991), by hormones (Lee et al., 1981; Hynes et al., 1981; Klock et al., 1987; Israël et al., 1989), by interferon (Hug et al., 1998; Arnheiter et al., 1990). Mention should also be made of the elements of inducible prokaryotic expression such as the Lac repressor system (LacR / operator / inducer) of E. coli (Hu et al., 1987; Brown et al., 1987; Labow et al., 1990), the tetracycline resistance system of E. coli (Gossen et al., 1992) (WO 94 04 672, EP 804,565).

These promoters may or may not be heterologous to the organism, may or may not be naturally present in the genome of the organism. Thus, they can be of viral origin.

Optionally, the marker qene and / or the selection gene and / or said third gene and / or the gene sequence of interest may be devoid of promoters or of expression elements and be placed under the control of a promoter or of endogenous expression elements of the retrovirus, such as for example the expression elements present in the LTRs. It is obvious that, depending on the desired result, those skilled in the art will choose and adapt the elements for regulating gene expression. Thus, when the retroviral vector according to the invention contains several genes to be expressed, it may be advantageous to use splicing signals, or to use IRES (Internai Ribosome Entry site) sequences; IRES sequences make it possible to translate several proteins from a single polycistronic RNA. Finally, it may also be interesting to have at least one retrovirus gene under control. an endogenous promoter, and at least one other gene under the control of a heterologous promoter.

To date, numerous proviral constructs have been successfully used to express genes of interest (for review, see Coffin JM, Hughes SH and Varmus HE, "Retrovirus", EDS. Cold Spring Harbor Laboratory Press. (1997), chapter 9 "Development and applications of retroviral vectors", pages 437-474.

The recombinant DNA technologies used for the construction of the retroviral vector according to the invention are those known and commonly used by those skilled in the art. Standard techniques are used for cloning, DNA isolation, amplification, and purification; enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases are carried out according to the manufacturer's recommendations. These and other techniques are generally performed according to Sambrook et al. (1989).

The gene of interest according to the invention codes for a gene product which has a particular function, for example which acts as a marker to produce a detectable product or, preferably, it is a therapeutic gene whose product is active against infection or disease. Thus the therapeutic genes can code for example for an antisense RNA, a ribozyme or a peptide or a protein such as a trans-dominant negative mutant of a target protein, a toxin, a conditional toxin, an antigen which induces an antibody or which induces T-helper or cytotoxic cells, a single chain antibody or a tumor suppressor protein. The gene of interest can also code for killer proteins as defined above. In the retroviral vector according to the invention, one or more genes of interest may be present. More generally, the present invention also relates to a viral vector, in particular a retroviral vector, characterized in that it contains at least one selection gene outside the packaging genome. Such a retroviral vector increases the percentage of cell clones producing recombinant viruses obtained after transfection of the packaging cells compared to the rate obtained with a viral vector whose selection gene is located in the packaging viral genome, and this, whatever the rate of drift of the packaging line. When it is a retroviral vector according to the invention, the selection gene is located outside of the LTR5 'and LTR3' sequences. The viral vector may also contain in its packaging genome one or more genes chosen from the group of genes of interest, marker genes, selection genes, so-called third genes according to the invention. These genes are under the control of elements of expression as defined above. Such a retroviral vector can be used to produce non-replicative retroviral particles for use in basic research but also for the preparation of a medicament intended for preventive and curative therapies. Such a vector can be prepared according to conventional techniques of recombinant DNA available to those skilled in the art. It is obvious that this type of vector can be used for the preparation of various packaged viruses such as, for example, adenoviruses and adenovirus-associated.

Other characteristics and advantages of the present invention will be better demonstrated on reading the following examples.

In these examples, reference is made to the following figures: Figure 1: Schematic representation of the SAITK vector (SVN-ADH-IRES-TK:: Sh Ble)

LTR: "Long Terminal repeat"; ADH: nucleotide sequence coding for Drosophila alcohol dehydrogenase; IRES 1518: internal ribosome binding site; TK :: Sh Ble: gene for fusion between the herpes simplex virus thymidine kinase and the phleomycin resistance gene. Figure 2: Schematic representation of the SASS BLE vector (SNV-ADH / SV40-Sh Ble)

The restriction sites Sspl at position 3 and Nde I at position 2728 are indicated; LTR: "Long terminal repea t"; ADH: nucleotide sequence coding for Drosophila alcohol dehydrogenase; Prom SV40: promoter of SV40; Sh Ble: phleomycin resistance gene; Poly A-SV40: polyadenylation sequence of SV40.

EXAMPLES

EXAMPLE 1: MATERIAL AND METHODS

1.1. Cell culture and infection

The QT6 and DSH-134G cell lines are grown under standard conditions. Briefly, the cells of the QT6 cell line derived from Japanese quail (Moscovici et al., 1977) are cultured in Ham's F10 medium (GIBCO-BRL-Life Technologies) supplemented with 10% tryptose phosphate broth (GIBCO- BRL-Life Technologies), 5% newborn calf serum (EUROBIO), 1% chicken serum (GIBCO-BRL-Life Technologies) and 1% penicillin-streptomycin (10,000 units per ml, GIBCO-BRL - Life Technologies). The DSH-134G packaging cell line derived from canine osteosarcoma (Martinez and Dornburg, 1995) is cultured in a DMEM medium containing 1 g / 1 D-glucose, 3.6% NaHC0 ₃ supplemented with 6% fetal calf serum (EUROBIO), 1% glutamine (GIBCO-BRL- Life Technologies) and 1% penicillin-streptomycin (10 000 units / ml, GIBCO-BRL-Life Technologies). For the transfections, the cells are seeded the previous day in a six-well plate at the rate of 10 ⁵ cells per well (COSTAR). Plasmids

(usually 5 μg) are introduced into the cells using the Transfectam® reagent (PROMEGA) according to the manufacturer's recommendations, except that 250 μl of transfection mixture are used per well instead of 500 μl.

The plasmids are introduced into QT6 or D17 cells in which the constructs are expressed under the control of the CMV promoter (cytomegalovirus). The polyclonal or clonal cell populations are selected in the presence of 5 μg / ml of phleomycin (CAYLA).

To generate the cell lines producing the SNV-ADH-IRES-TK retroviral vectors:: Sh ble, the SNV constructs are transfected into DSH 134G cells and selected with 50 μg / ml of phleomycin. When the colonies reach approximately one hundred cells, the cell clones contained in the culture dishes are scraped off and aspirated under a microscope with a pipette type P200 of GILSON®; cells are transferred to 48-well culture dishes (COSTAR) and then screened for the presence of retroviral particles.

In order to determine the viral titer, QT6 cells are seeded in six-well plates, 24 hours before infection (2 x 10 ⁵ cells / well) and infected with serial dilutions (decimal) of cell media not containing virus. Two days later, the infected cells are washed twice with PBS supplemented with Ca ²⁺ and Mg ²⁺ , then fixed for five minutes with 2% formaldehyde (MERCK-CLEVENOT) / 0.1% glutaraldehyde (SERVA), then washed with PBS supplemented with Ca ²⁺ and Mg ²⁺ and used in an ADH test. 1.2. Harvesting viruses and infections

Harvesting and infection protocols are based on those described by Gautier et al. (2000). The viruses are harvested from almost confluent packaging cells in a monolayer 20 to 24 hours after changing the medium. The harvested medium is centrifuged at 5,000 rpm for ten minutes and the virus is then titrated on QT6 cells. Briefly, the day before the titration, 5 x 10 ⁵ QT6 cells are seeded on six-well plates. The cells are infected with decimal dilutions of the viral supernatant and then returned to the incubator for an additional 48 hours. The cells are then fixed and stained for the ADH test. 1.3. ADH test Two days later, the infected cells are washed twice with PBS supplemented with Ca ²⁺ ions and

Mg ²⁺ , fixed for five minutes with 2% formaldehyde

(MERCK-CLEVENOT) / 0.1% glutaraldehyde (SERVA), then washed in PBS supplemented with Ca ²⁺ and Mg ²⁺ . After fixing and rinsing, the cells are covered with a minimum volume of the staining solution which consists for 1 ml in 900 μl of 0.15M Tris pH 8.5, 70 μl of a solution of Nicotina ide Adenine Dinucleotide (NAD) at 10 mg / ml in water (ICN), 22 μl of 2-butanol (PROLABO), 11 μl of phenazine methosulfate at 0.5 mg / ml of distilled water (SIGMA) and 10 μl of TetraNitroBlue Tetrazolium (TNBT ) (JANSEN CHEMICA), 10 mg in 700 μl of dimethylformamide + 300 μl of water); the cells are then incubated in the dark for six to twelve hours at room temperature. 1.4. Expression vectors and retroviral constructs

1.4.1. SNV-TK:: Sh ble vector The vector is constructed using an SNV-based vector containing the Sh ble:: NLSlacZ gene (Jaffredo et al., 2000). The vector is digested with HindIII; the cohesive ends are made blunt. The linear vector is then digested with Ncol and the vector fragment is gel-purified. The TK :: Sh ble fragment is removed from the put 102 vector (CAYLA) by digestion with SalI. The ends of the TK :: Sh ble fragment are made blunt then this fragment is digested with Nco I. The 1.55 kb fragment is then purified on gel and then ligated into the SNV vector prepared as described above.

1.4.2. SNV-IRES-Sh ble vector :: lacZ

The vector is constructed using an SNV-based vector containing the Sh ble :: lacZ fragment of the SNV gene.

(Jaffredo et al., 2000). The vector is then digested with Ncol and Xbal. IRES 1518 is inserted using the restriction enzymes Xbal and Ncol.

1.4.3. Vector SNV-ADH-IRES-Sh ble :: lacZ

The vector is constructed using an SNV-IRES-Sh ble :: lacZ fragment opened by Xbal. The 0.8 kb ADH gene is removed from the TA cloning vector (Gautier et al., 1996) by digestion with Spel / Xbal then purified on gel and finally ligated into the vector SNV-IRES-Sh ble :: lacZ.

1.4.4. SNV-ADH-IRES-TK:: Sh ble vector (SAITK vector) The SNV-ADH-IRES-TK:: Sh ble vector, also called SAITK, is constructed using the previously digested SNV-Tk :: Sh ble fragment by Ncol / Pmll enzymes. The 1.5 kb ADH-IRES gene is removed from the SNV-ADH-IRES- Sh ble :: lacZ vector by Ncol / Smal digestion and then purified on gel and finally ligated into the vector SNV-TK:: Sh ble.

1.5. Elimination of non-interfering cells from culture DSH 134G or DSH cells producing SNV-Sh-ble:: NLS lacZ are seeded at low density in duplicate in six-well dishes. The cells are infected twice overnight at one day apart using 1 ml of SAITK viral supernatant (2 × 1.5 10 ⁶ AFU) in the presence of 2 μg / ml of polybrene. Two days later, the DNA expression is revealed on one of the six-well dishes while the second dish receives 100 μMole of acyclovir per well. Selection with acyclovir is complete after eight days. The culture medium is then removed and replaced with fresh medium without acyclovir. The cell clones resistant to acyclovir are then tested for their ability to produce viruses. EXAMPLE 2: CHARACTERIZATION OF THE DERIVATIVE 2.1. Drift estimation The more a packaging line drifts, the less retroviruses it produces and the more it becomes infectious with the retroviruses it is supposed to produce. This phenomenon, which can be highlighted during transfections of vectors equipped with a selection gene, is responsible for obtaining a number of producer clones recovered which may drop below 1% (the 99% of clones not mostly resulting from virus infected cells from the few transfected producer cells). It therefore seems important, when one wants to work clonally, to know the speed of drift of the line used. For this, the inventors infected the packaging line DSH134G with a retrovirus produced by this line and provided with a reporter gene. 48 hours after infection, it appears that the drift is proportional to the number of infections found. An infection carried out with the vector SNV-lacZ (also called CXL) (suspension of 5.10 ⁵ viral particles per ml) on DSH-134G cells, after numerous passages in culture, allowed the inventors to reach a level of infectable cells exceeding 50%.

With the DSH 134G, the drift phenomenon seems to be constant. In fact, when the line has undergone a correction of its drift, there is a reappearance of the infectable cells.

(PV: viral particle; DSH-PL: DSH 134G packaging line transfected with an SNV-phleomycin-LacZ vector)

2.2. Infectible cells do not produce viruses DSH-CXL cells, which correspond to the DSH-134G line transfected with an SNV-lacZ vector, seeded at low density, are infected with the SAITK vector and then selected by Phleomycin. Resistant clones appear and cells are tested for their ability to produce viral particles. No clone produced viral particles. This demonstrates that permissive cells have lost their production capacity. 2.3. Suicide vector

The inventors have thought of eliminating these infectious cells which do not produce viruses by infecting them with an SNV vector carrying a suicide gene. The inventors have built a three-way vector called SAITK

(figure 1) which is composed as follows:

- a reporter gene which is Drosophila Alcohol DeHydrogenase (ADH), which makes it possible to know easily, by histochemical reaction, the viral titer of the different clones selected but also to check whether the cells to be eliminated have been completely destroyed by the absence of positive ADH cells in the "corrected" packaging line;

- an Internal Ribosome Linking Site (IRES) which allows balanced expression of the two genetic units of the vector.

- A TK :: ShBle fusion gene which includes the suicide function carried by the Thymidine Kinase (TK) gene in the presence of Acyclovir and that of resistance to Phleomycin (ShBle) which allows the rapid selection of producer clones. The cells expressing the vector can therefore be either killed or positively selected. It should be noted that this suicide gene cannot be used on cells which have been transfected with the dihydrofolate reductase gene which confers resistance to methotrexate because Acyclovir kills the cells which possess it.

This SAITK vector was produced by transfection into the packaging line DSH-134G. The inventors have recovered two of the 93 producer clones tested.

2.4. Effect of Acyclovir on Retroviral Production DSH134G Cells Producing an SNV-lacZ Vector

(also called DSH-CXL or CXL cells) were seeded in six-well plates and brought into contact with increasing doses of Acyclovir (0, 50 and 100 μM / well). Tests for the production of viral particles were carried out over time for the three concentrations. For the first two weeks, the cells remained in the presence of Acyclovir. The latter was then removed from the medium.

The effect of Acyclovir, although strongly decreasing the production of viral particles during treatment, has a reversible effect.

EXAMPLE 3: DERIVATIVE CORRECTION

3.1. Elimination of permissive cells by a virus carrying the Thymidine Kinase gene. The SAITK virus suspension (1.5 10 ⁶ PV / ml) was deposited on the naive DSH-CXL or DSH-134G cells

(DSH 134G cells having never been transfected with a retroviral vector) previously seeded at very low density in three wells (six-well plate). 48 hours later, the cells are treated with Acyclovir phosphoryl (ZOVIRAX®) at a concentration of 100 μMolar for ten days.

- In the three wells, only the interfering cells are selected by Zovirax and constitute a line enriched in potentially producing cells.

- In the first well, the cell culture is amplified. - On the two remaining wells, checks for the presence of ADH cells positive or resistant to phleomycin were carried out. They turned out to be negative. 3.2. Elimination of non-interfering cells DSH 134G cells are seeded at low density in duplicates in six-well plates. Then, the cells are infected for two consecutive nights with 1 ml of SAITK supernatant (2 × 1.5 × 10 ⁶ PV / ml) in the presence of 4 μg of polybrene in a final volume of 2 ml. After two days, the presence of viral particles is checked by revealing the DHA on one of the plates and on the other plate the cells are treated with 100 μMol of Acyclovir (ZOVIRAX). In seven to ten days, the selection is made and the cells are returned to normal medium to allow the interfering clones to grow more easily.

The surviving DSH-134Gs are tested for their capacity for packaging after transfection of SNV vectors. For untreated cells, the inventors obtain a maximum of 6% of producer clones. After treatment, the results go up to 85%.

EXAMPLE 4: CREATION OF A NEW MULTIFUNCTIONAL VECTOR: SASSHBLE (SNV, ADH, SV 40, Phleomycin)

The transfection results obtained show that the vectors containing a selection gene inside the encapsidable viral genome induce a number of selected clones much greater than the number of clones actually transfected. This phenomenon is particularly true when the packaging line has drifted. Two vectors can be co-transfected, one containing the genome encapsidable and the other, non-encapsidable carrying a selection gene, the cells which have received the selection plasmid are recovered but among them, only part received the encapsidable vector. The number of clones to be tested therefore remains, in both cases, significant. The inventors have hypothesized that the majority of non-producing clones originate from the infection of non-interfering cells by viruses produced by transfected cells. The inventors have imagined dissociating the viral genome from the selection gene while preserving the two on the same plasmid (FIG. 2).

The comparison between the vector SASSHBLE and the vector SNV 818 (Gautier et al., 1996) is given in the table below. These two vectors have the Alcohol Dehydrogenase gene and the phleomycin resistance gene, but SNV 818 has ADH and phleomycin fused on the same genetic unit and under the control of SNV LTRs.

These results were obtained on DSH-134Gs not treated with the SAITK-Acyclovir system.

In view of these results, the inventors have imagined combining the use of this type of vector and the treatment with SAITK-Acyclovir.

After transfection into DSH-134G, the cells are selected by phleomycin, then the titration is carried out as soon as the cells are ready.

(DSH-GAR: DSH 134G "control" cells, resistant to Acyclovir)

The inventors confirmed this result at the clonal level; the vectors used are the same as above.

It is noted, for the SNV 818 type vectors, that the percentage of producer clones goes from 6% for the untreated cells to 85% for the treated cells. The percentage of interfering clones is even more eloquent since it goes from 7% for untreated cells to 100% for treated cells.

For SASSHBLE type vectors, the results go in the same direction (even if the percentage of producer clones before treatment is more than four times greater), the percentage after treatment is of the same order of magnitude and reaches 79% with a rate interference from 22 to 95%.

These results indicate to the inventors that for DSH 134G, the maximum ratio of producing clones is of the order of 4/5 after transfection and that the 1/5 ^th not producing, seems a constant ratio. This seems to be due to the location of the integration of the transfected vector in the genomic DNA of the cell. This integration site is incompatible with the production of viral particles.

The inventors have thus shown that, after numerous passages, the packaging cells DSH 134G tend to lose their capacity to produce viral particles. Many of them then become permissive and can be infected with viruses of the same type. The treatment of DSH-134G with the SAITK-Acyclovir system makes it possible to eliminate the majority of the cells which were permissive and therefore non-productive. This treatment must be prior to transfection. It allowed the inventors to obtain between 79 and 85% of producer clones. In parallel, the use of a vector such as SASSHBLE makes it possible to obtain, after transfection, the maximum percentage of producer clones available whatever the rate of drift of the packaging line.

It should be noted that, alternatively, the vector SAITK can be substituted for a vector SNV-GFP and, after infection, subject the cells to sorting with FACS, in order to allow the line to be enriched more quickly. encapsidation in interfering cells and avoid treatment with Alcyclovir.

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Claims

1. Process for obtaining retrovirus packaging cells, trans-complementants non-permissive to infection by said retrovirus, characterized in that it comprises the steps of: (i) culturing a population of said packaging cells; (ii) introduction by retroviral infection into the said cells cultivated in (i) of a retroviral vector comprising at least one “marker” gene included in the encapsidable retroviral genome; (iii) selection of said cells not expressing said “marker” gene. 2. Method according to claim 1, characterized in that said selection is carried out by eliminating, from said population of packaging cells, the cells expressing said “marker” gene. 3. Method according to claim 2, characterized in that said “marker” gene is a “suicide” gene whose cellular expression causes the death of the cells expressing said “suicide” gene. 4. Method according to claim 3, characterized in that it further comprises the step of administering in the culture medium a pro-drug in an amount sufficient for said pro-drug after having been metabolized by the product of expression of said “suicide” gene is converted into a compound toxic to said cell expressing said “marker” gene. 5. Method according to claim 3, characterized in that said “suicide” gene codes for a protein chosen from the group consisting of kinases, bacterial cytosine deaminase, hypoxanthine phosphoribosyl transferase (HPRT), guanine phosphoribosyl transferase (GpT). 6. Method according to claim 5, characterized in that said kinase is chosen from the group composed of thymidine kinase of the Herpes simplex virus, of thymidine kinase of chickenpox virus. 7. Method according to claim 6, characterized in that said kinase is the thymidine kinase of the herpes simplex virus and the pro-drug is chosen from the group composed of acyclovir, ganciclovir, FIAU or their derivatives . 8. Method according to claim 6, characterized in that said kinase is the thymidine kinase of the chickenpox virus and the pro-drug is chosen from the group composed of 6-methoxypurine arabinoside or one of its derivatives. 9. Method according to claim 5, characterized in that said protein expressed from said suicide gene is bacterial cytosine deaminase and the prodrug is chosen from the group composed of 5-fluorocytosine and its derivatives. 10. The method of claim 5, characterized in that said protein expressed from said suicide gene is hypoxanthine phosphoribosyl transferase (HPRT) and the prodrug is chosen from 6-thio-guanine and its derivatives. 11. Method according to claim 5, characterized in that said protein expressed from said suicide gene is guanine-phosphoribosyl-transferase (GpT) and the prodrug is chosen from 6-thio-xanthine and its derivatives. 12. Method according to claim 2, characterized in that said “marker” gene is a “killer” gene which codes for a pro-apoptotic protein whose cellular expression causes the death of the cells expressing said “marker” gene. 13. The method of claim 12, characterized in that said pro-apoptotic protein is chosen from preferably among the proteins Bcl2, BIK, BAK, BID, BAX, the caspases, the AIF protein, TNF and the proteins of the TNF family, the FASL protein. 14. Method according to claim 1, characterized in that said selection is carried out by sorting in said population of packaging cells the cells which express and which do not express said marker gene. 15. The method of claim 14, characterized in that said marker gene is a "reporter" gene. 16. Method according to claim 15, characterized in that said “reporter” gene codes for an autofluorescent protein chosen from the group composed of green fluorescence protein (GFP), red fluorescence protein (RFP), and variants fluorescent of these proteins. 17. The method of claim 15, characterized in that said “reporter” gene codes for an enzyme detectable by a histochemical process on living cells. 18. The method of claim 17, characterized in that said enzyme is selected from the group consisting of β-galactosidase, β-glucoronidase, alkaline phosphatase, alcoholic dehydrogenase, luciferase, chloramphenicol- acetyl transferase. 19. The method of claims 14 to 18, characterized in that it further comprises the step of sorting the population of packaging cells by means of a cell sorter. 20. Method according to any one of the preceding claims, characterized in that said retroviral vector further comprises a second selection gene. 21. The method of claim 20, characterized in that said second gene for selection of said vector retroviral is located outside the encapsidable retroviral genome. 22. Method according to claims 20 and 21, characterized in that said second selection gene is chosen from antibiotic resistance genes. 23. The method as claimed in claim 22, characterized in that said antibiotic is chosen from the group consisting of neomycin, tetracycline, ampicillin, kanamycin, phleomycin, bleomycin, hygromycin, chloramphenicol, carbenicillin, geneticin. 24. Method according to any one of the preceding claims, characterized in that said retroviral vector further comprises a third gene coding for an enzyme located inside the encapsidable retroviral genome. 25. The method of claim 24, characterized in that said method further comprises a step of titrating the viral particles by assaying the activity of said enzyme. 26. The method of claim 25, characterized in that said enzyme is alcohol dehydrogenase. 27. Method according to any one of the preceding claims, characterized in that said retrovirus is chosen from the group consisting of ALV, AEV, AMV, RSV, RAV, MMTV, SNV, Mo-MLV, Fe-LV, BLV, HTLV -1, HTLV-2, HIV-1, HIV-2, SIV, FIV, BIV. 28. The method of claim 27 characterized in that said retrovirus is SNV. 29. Use of retroviral vector for the implementation of a process for obtaining non-permissive trans-complementing retrovirus packaging cells, characterized in that said vector comprises a marker gene chosen from the group consisting of suicide genes , killer genes, "reporter" genes located between the LTR5 'and LTR3' sequences. 30. Use according to claim 29, characterized in that said retroviral vector derives from a retrovirus chosen from the group composed of ALV, AEV, AMV, RSV, RAV, MMTV, SNV, Mo-MLV, Fe-LV, BLV, HTLV-1, HTLV-2, HIV-1, HIV-2, SIV, FIV, BIV. 31. Use according to claims 29 to 30, characterized in that the expression of said marker gene is under the control of a retroviral promoter located in the LTR sequence. 32. Use according to claims 29 to 31, characterized in that said suicide gene is chosen from the group composed of the genes of thymidine kinase from the Herpes simplex virus, from thymidine kinase from chickenpox virus, from cytosine deaminase bacterial, hypoxanthine phosphoribosyl transferase (HPRT), guanine phosphorybosyl transferase (GpT). 33. Use according to claim 29 to 31, characterized in that said killer gene is chosen from the genes of pro-apoptotic proteins composed of the proteins Bcl2, BIK, BAK, BID, BAX, caspases, AIF protein, TNF and TNF family proteins, FASL protein. 34. Use according to claims 29 to 31, characterized in that said “reporter” gene codes for an autofluorescent protein chosen from the group consisting of green fluorescence protein (GFP), red fluorescence protein (RFP), and fluorescent variants of these proteins. 35. Use according to claims 29 to 31, characterized in that said “reporter” gene codes for an enzyme detectable by a histochemical process on living cells, said enzyme being chosen from the group composed of β-galactosidase, of β - glucoronidase, alkaline phosphatase, alcoholic dehydrogenase, luciferase, chloramphenicol-acetyl transferase. 36. Use according to claims 29 to 35 characterized in that said vector further comprises a said second selection gene. 37. Use according to claim 36, characterized in that said second selection gene is located outside the encapsidable retroviral genome. 38. Use according to claims 36 to 37, characterized in that said second selection gene is chosen from antibiotic resistance genes, said antibiotic being chosen from the group consisting of neomycin, tetracycline, ampicillin, kanamycin , phleomycin, bleomycin, hygromycin, chloramphenicol, carbenicillin, geneticin. 39. Use according to any one of claims 29 to 38, characterized in that said retroviral vector further comprises a said third gene coding for an enzyme and located between the sequences LTR5 'and LTR3'. 40. Method for preparing a non-replicating recombinant retrovirus, characterized in that said method comprises the following steps: (i) obtaining, by implementing a method according to claims 1 to 28, trans-complementary non-permissive packaging cells; (ii) transfection into said cells obtained in (i) of a retroviral vector comprising at least the encapsidable genome of said recombinant retrovirus; (iϋ) culture of the cells thus transfected under conditions allowing the expression of the packaging proteins; (iv) harvesting of said recombinant retroviruses. 41. Method according to claim 40, characterized in that the genome of said recombinant retrovirus contains at least one sequence coding for a protein of interest. 42. Method according to claims 40 and 41, characterized in that said retroviral vector comprises at least one positive selection gene. 43. The method according to claim 42, characterized in that said selection gene is located outside the encapsidable retroviral genome. 44. Method according to claims 42 and 43, characterized in that said selection gene is a gene for resistance to an antibiotic and that said culture of step iii) is carried out in the presence of said antibiotic.