FR2956409A1 - METHODS OF OPTIMIZING VIRAL PRODUCTION BY INACTIVATION OR INHIBITION OF HIPK2 GENE AND MODIFIED CELLS FOR VIRAL PRODUCTION - Google Patents

Abstract

Improvement of virus production in cell culture by suppression or inhibition of the HIPK2 gene in infected cells.

Description

Methods for optimizing virus production by inactivating or inhibiting the HIPK2 gene and cells modified for viral production

The present invention provides methods and cells for enhancing the production of virus and viral proteins in cell culture. In particular, the invention relates to improving the industrial production of vaccine seeds or influenza virus antigens in cell culture. For the production of vaccine seeds, influenza viruses are typically produced in chicken eggs. This production of influenza viruses in eggs is not without many problems including the time required for the reassortment of viruses and the production of vaccine seeds. In addition, the poultry itself is likely to be affected by influenza viruses which could cause supply difficulties if farms intended for virus production were themselves affected by an influenza virus pandemic.

A first alternative would be the production of viruses in cell cultures, this solution is also used for many viruses including influenza viruses. From virus produced in cell culture, viral antigens can be purified according to standard techniques. Another solution would be the direct production of viral antigens in cell culture. However, both the production of viruses and the production of viral antigens in cell culture encounter difficulties in terms of yield and thus of the amount of virus or antigen produced. In the particular case of influenza viruses, cell lines with regulatory approvals such as MDCK, Vero, BHK21, CHO, HEK 293 and PERCE are not optimized for vaccine seed production. These lines do not make it possible to obtain adequate production yields for manufacturers. It is the same for other viruses such as adenovirus or herpes simplex virus (HSV). The research therefore focuses on the development of new cell lines for the production of viruses in cell culture as well as on the improvement of the yields of viral and virus production in cell lines available for industrial production. HIPK2 (Homeodomain-interacting protein kinase 2) is a serine-threonine kinase protein originally identified as a co-repressor of homeo- domain transcription factors. Since then, HIPK2 has been the best characterized member of the HIPK (1 to 4) protein family; it is a co-regulator that phosphorylates many factors and cofactors of cellular transcription. HIPK2 is activated in response to DNA damage agents and morphogenetic signals. It is therefore involved in the regulation of gene expression programs for cell differentiation, development and apoptosis. HIPK2 has a pleiotropic effect and is involved in many cellular pathways, including p53. The stable Knockout of HIPK2 in MCF7 cells (breast cancer), for example, induces misfolding of p53 and inhibition of the trancription of p53 target genes. HIPK2 has therefore been the subject of research in the field of oncology. In addition, WO 2004/108754 as well as Giraud et al. disclose that the US 11 protein of Herpes Simplex Virus (HSV) interacts with HIPK2. No effect on virus replication, viral production or viral antigen production has however been reported. Unexpectedly, the present invention shows that suppressing HIPK2 expression in producer cells provides better replication and viral production. The methods of the present invention thus make it possible to improve the production of viruses and viral antigens in existing cell lines by inactivating or inhibiting the expression of the HIPK2 gene in these cells. Thus, the present invention also relates to novel cells and cell lines particularly suitable for viral production in which the HIPK2 gene has been inactivated or inhibited. One of the applications of the methods and modified cells of the present invention is the production of influenza viruses for use as vaccine seeds. The aim is to achieve yields that are similar to or better than the yields achieved by current chicken egg production processes or current production processes on regulated cell lines.

SUMMARY OF THE INVENTION The invention thus relates to methods of in vitro production of virus by cell culture comprising the infection of cells with a virus and the replication of the virus in these cells wherein the cells are modified cells in which the HIPK2 gene is inactivated or in which the expression of the HIPK2 gene is inhibited.

Preferably, the methods according to the invention further comprise isolating the viruses from the cell culture. In a first embodiment, the modified cells have a deletion of the HIPK2 gene.

In a second embodiment, the modified cells express an interfering RNA specific for HIPK2. Preferably, the invention relates to methods for producing viruses selected from adenoviruses, HSV viruses and viruses of the family of orthomyxoviridae. More preferably, the invention relates to methods of producing viruses selected from influenza viruses of type A, B and C. Advantageously, the cells are selected from mammalian cells, avian cells or insect cells. Preferably, the cells are chosen from CHO, MDCK, COS, CV1, Vero, BHK21, PERC6, HEK, HeLa, AGEI.CR, AGEI.CR.pIX, EB66, EBx and HEp-2 cells. The invention also relates to the production of viral products comprising the in vitro production of virus by cell culture according to one of the preceding claims. The subject of the invention is also cells modified for the production of virus by cell culture in which the HIPK2 gene is inactivated or the expression of the HIPK2 gene is inhibited; these cells being infected by a virus. The subject of the invention is also cells modified for the production of virus by cell culture chosen from CHO, MDCK, COS, CV1, Vero, BHK21, PERC6, HEK, HeLa, AGEI.CR and AGEI.CR.pIX cells. EB66, EBx and HEp-2; in which the HIPK2 gene is inactivated or the expression of the HIPK2 gene is inhibited. Preferably, the cells modified according to the invention are infected with a virus, preferably with a virus chosen from adenoviruses, HSV viruses and viruses of the ortyhomyoxoviridae family, more preferably by a virus chosen from influenza A viruses. , B or C. In a first embodiment, the HIPK2 gene is inhibited by the expression of a HIPK2-specific interfering RNA. In a second embodiment, the HIPK2 gene has been deleted.

DESCRIPTION OF THE INVENTION

The invention thus relates to a method of producing virus by cell culture comprising the infection of cells with a virus and the replication of the virus in these cells wherein the cells are cells modified in such a way that the HIPK2 gene is inactivated or such that the expression of the HIPK2 gene is inhibited. The invention also relates to a method for producing viral antigens by cell culture comprising expressing the viral antigens in the cells wherein the cells are cells modified such that the HIPK2 gene is inactivated or in such a way that the HIPK2 gene expression is inhibited. The methods of the present invention are preferably in vitro methods. The invention thus particularly relates to a method of producing virus by cell culture comprising infecting the cells with a virus and culturing the cells to obtain viral replication. Following virus replication, the method may include isolating or purifying viruses produced from the cells or cell culture. By "isolation or purification of viruses" is also meant the production and purification of viral products including viral antigens. Cell culture, infection of the cells with a virus and viral replication in the cells are carried out according to the usual techniques in this field.

The present invention is based on the unexpected observation that inactivation or inhibition of the HIPK2 gene in cells enhances replication or virus production in cells. Thus, the culture of modified cells in which the HIPK2 gene is inactivated or in which the expression of the HIPK2 gene is inhibited, makes it possible to significantly increase the yield of virus production by cell culture.

These modified cells are also useful for increased production of viral antigens by direct expression of genes encoding these antigens in cells. The modified cells used in the methods according to the invention have, for example, a deletion of the HIPK2 gene or they express a HIPK2-specific interfering RNA that inhibits the expression of this gene in the cells. These cells are used for the production of viruses or for the direct production of viral antigens. The cells modified according to the present invention are useful for the industrial production of viruses, viral products, vaccine seeds and / or viral antigens. In preferred embodiments, the modified cells are thus infected with a virus and allow the multiplication or replication of the virus in the cell. Preferably, the virus is a virus selected from adenoviruses, HSV viruses and viruses of the family of orthomyxoviridae, more preferentially, the virus is chosen from influenza viruses of type A, B and C. The modified cells used in the methods of the present invention as well as the modified cells of the present invention may be any type of cell suitable for cell culture, replication, virus production and viral antigen production.

Thus, the cells are for example selected from mammalian cells, avian cells and insect cells. Among the mammalian cells, mention will be made in particular of culture cells derived from human cells, pig or dog cells. These cells can in particular be derived from pulmonary epithelium cells (eg human lung carcinoma human A549 CCL-185) or intestinal epithelium (Caco-2 ATCC HTB-37). Among the mammalian cells, mention may also be made of the following cells: o SJPL pig lung epithelium cells including, in particular, the ATCC-deposited line PTA-3256 (US2004 / 0142450 and WO2009 / 059411), or MDCK cells of dog, o BHK21 CCL-10 Mesocricetus auratus (hamster), o Human HOS cells including the CRL-1543 line. Among the insect cells, mention will in particular be made of Drosophila cells and cells selected from SF9 cells CRL-1711, Schneider's Drosophila Line 2 CRL-1963 and BM-N CRL-8910 bombyx.

In a preferred embodiment, the cells are avian cells adapted to cell culture such as for example: CCL-141 Anas platyrhynchus domesticus, CRL-12135 Gallus gallus Con A-Cl VICK, CRL-12203 Gallus gallus UMNSAH / DF-1, o CRL-12357 Gallus gallus ConA-B1-VICK, o CRL-1590 Gallus gallus SL-29, or CRL-1708 Coturnix coturnix japonica QT6, or CRL-1962 Coturnix coturnix japonica QM7 (Quail muscle clone 7 ), o CRL-2111 Gallus gallus (chicken) DT40, o CRL-2112 Gallus gallus (chicken) DT95, o CRL-8135 Meleagris gallopavo (turkey) MDTC-RP19, and o and the QM7 line of myoblasts deposited at ATCC (CRL-1632) described in US2005 / 0084503.

Other avian cell lines are known to those skilled in the art and can be used. It may in particular be avian embryonic cells. The EB66 and EBx cell lines have been described in WO03 / 076601 and WO2008129058. Lohr et al. describe the establishment of avian cell lines AGEI.CR and AGEI.CR.pIX for viral propagation or replication. WO2009 / 004016 also describes immortalized avian cells for viral production and methods for their preparation. Preferably, these cells are chosen from MDCK, Vero, A549 cells and, generally, from avian cell lines used for the production of influenza viruses. MDCK cells are for example described in US2006 / 0188977 and EP1862537. In particular, the following lines are mentioned: MDCK-SF101 (ATCC PTA-6501), MDCK-SF102 (ATCC PTA-6502) and MDCK-SF103 (ATCC PTA-6503). The cells are preferably chosen from the cells: o Vero cells, in particular the CCL-81 line Cercopithecus aethiops Vero; o CHO cells, in particular lines CRL-12444 Cricetulus griseus (Chinese hamster) and CHO DP-12 clone # 1933 aIL8.92 NB 28605/12; o MDCK cells, in particular the CCL-34 line Canis familiaris MDCK (NBL-2), the COS cells in particular the CRL-1650 Cercopithecus aethiops lines, COS-1 CRL-1651 Cercopithecus aethiops and COS-7 lines; the CV1 cells, in particular the CCL-70 Cercopithecus aethiops CV-1 lines, the HeLa cells, and the HEp-2 cells. Advantageously, the cells are chosen from cell lines benefiting from regulatory authorizations for viral production in cell culture for medical purposes. Preferably, the cells are chosen from BHK21, Vero, PERCE, HEK and EB66 cells.

In advantageous embodiments, the cells are chosen from CHO, MDCK, COS, CV-1, Vero, BHK21, PERCE, HEK, HeLa, AGEI.CR, AGEI.CR.pIX, EB66, EBx and HEp-2. The use of CV-1, CV-C, Vero and BSC-1 cell lines for viral production is for example described in US 6,303,371. The cells of the present invention are modified cells. By "modified cell" is meant a cell whose genome has been modified or into which extra-chromosomal genetic material has been introduced. Typically, this change is a stable or constitutive change. By "modified cell" is also meant a cell in which the HIPK2 protein is inactivated or inhibited. Preferably, in the cells modified according to the present invention, the HIPK2 gene is inactivated or the expression of the HIPK2 gene is inhibited. The inactivation or knock-out of the HIPK2 gene can be achieved by any method of site-specific mutagenesis or recombination known to those skilled in the art. In preferred embodiments, the cells have been modified by the deletion of the HIPK2 gene. The gene may also be inactivated by the insertion of a sequence into the HIPK2 gene that interrupts the open reading frame of the HIPK2 gene. When the HIPK2 gene is inactivated there is no longer any expression of the HIPK2 gene in the modified cell. Inactivation of the HIPK2 gene does not affect cell viability.

By "modified cell in which the expression of the HIPK2 gene is inhibited" is meant modified cells in which the expression of the HIPK2 gene is reduced by at least 50%, 60%, 70%, 80%, 90%, 95% or 98% compared to the same unmodified cell. Inhibition of gene expression may affect transcription or translation of the HIPK2 gene. Alternatively, this inhibition is an inhibition of the activity of the HIPK2 protein. In a preferred embodiment, the HIPK2 gene is inhibited by the expression of a HIPK2-specific interfering RNA. The inhibition of the expression or the activity of the HIPK2 gene can be carried out by all the usual means available to those skilled in the art for controlling the expression of the genes that can be used, in particular the use of inhibitors. transcription or translation. In particular, the use of interfering RNA makes it possible to specifically inhibit the expression of genes of interest. The interfering RNA molecules are used to specifically target messenger RNAs (mRNA): the interfering RNA hybridizes to the mRNA and consequently inhibits the translation of the corresponding protein, either by simple steric hindrance or by promoting cleavage of the mRNA. The interfering RNA can be selected from several forms such as: antisense RNA, double-stranded RNA, "small interfering" RNA (siRNA), "Small Hairpin" RNA (shRNA) or ribosomal RNA. SiRNAs, for "Small Interfering RNA" or small interfering RNA, are short sequences of about 15 to 30 base pairs (bp), preferably 19 to 25 bp. They comprise a first strand and a complementary strand identical to the targeted region of the RNA of the target gene. SiRNA refers to synthetic double-stranded RNA forms. ShRNAs for "Small Hairpin RNA" are double-stranded RNAs produced by a sequence cloned into an expression vector, encoding an RNA molecule that will adopt a hairpin shape after cleavage. The design and preparation of interfering RNAs and their use for transfection of cells in vivo and in vitro are well known and widely described in numerous publications. SiRNAs can be designed and prepared using appropriate software available online. The tools for the preparation of siRNA and the transfection of cells are available to the public, for example the siRNA vectors marketed by Invitrogen®. By "interfering RNA specific for HIPK2" is meant a single or double-stranded ribonucleic acid, which interferes with the messenger RNA specific for the HIPK2 gene leading to its degradation and to the decrease in its translation into protein. In a preferred embodiment, the expression of the HIPK2 gene is inhibited by the modification of the cell to obtain the expression of a HIPK2-specific siRNA having the sequence "sense" 5'-GACACCAGAUGACCAUGAA-3 'and antisense 5' -UUCAUGGUCAUCUGGUGUC- 3 '. In another embodiment, the modified cells express a protein "neutralizing" the activity of HIPK2. For example, it may be a protein binding to HIPK2. In another embodiment, the modified cells express a negative dominant mutant of HIPK2. Any other suitable method may be used to inactivate or inhibit the HIPK2 gene in the modified cells.

The HIPK2 gene is a gene conserved within different species and the skilled person will know how to inactivate or inhibit the HIPK2 gene in the various cells used for viral production.

The table above lists the GENBANK references of the HIPK2 gene in different species. species symbol gene / UniGene RefSeq Accession Homo sapiens HIPK2 28996 NM_001113239 AAL37371 NM_022740 AAG41236 Mus musculus HIPK2 15258 NM_010433 AAK07649 NM_001136065 AAK07650 Rattus norvegicus HIPK2 362342 XM_001066812 XM_342662 Danio rerio HIPK2 557551 NM_001080160 BC171706 NM_001099985 Pan troglodytes HIPK2 463772 XM_519419 XM_001151817 Gallus gallus HIPK2 374138 XM_416335 AF461697 BX950640 Canis Thus, those skilled in the art have the information necessary for the inactivation or inhibition of the HIPK2 gene in cells derived from different species. In a preferred embodiment of the invention, the modified cells of the present invention are infected with a virus for the production of this virus by cell culture. Any type of virus that can replicate in in vitro cell cultures can be produced by the methods of the present invention. Advantageously, the viruses are chosen from DNA viruses, for example adenoviruses and HSV viruses. In another embodiment, the viruses are selected from RNA viruses such as viruses of the family Orthomyxoviridae and in particular influenza viruses type A, B and C humans and animals. Preferably, these are viruses having an interest in a vaccine application and more particularly influenza virus. Thus, the invention also relates to a method of producing a vaccine seed comprising the in vitro production of virus by cell culture according to the methods of the present invention or using cells modified according to the present invention.

FIGURES

Figure 1: Quantification of the production of A / H3N2 virus in a state of extinction of the endogenous expression of HIPK2 by transfection of siRNA (SiHIPK2). After transfection with SiRNA, HeLa cells were infected with A / H3N2 virus (at MOI (, 1) .The viral offspring produced at 8, 24 and 32 hpi was titrated with RT-qPCR. HIPK2 induces an increase in the amount of A / H3N2 virus produced relative to the production conditions in non-transfected (T) cells or transfected with a control siRNA (SiC) Figure 2: Quantification of Ad5 production under conditions of Endogenous Expression of HIPK2 by Transfection of siRNA (SiHIPK2) After transfection with SiRNA, HeLa cells were infected with Ad5 (at MOI 0.1 and 1. The viral offspring produced at 36 and at 90 hpi was titrated on 293 cells. The extinction of HIPK2 induced an increase in the amount of Ad5 produced relative to the production conditions in cells transfected with a control siRNA (SiC).

Figure 3: Quantification of the production of HSV-1 under conditions of extinction of the endogenous expression of HIPK2 by transfection of siRNA (SiHIPK2). After transfection with SiRNA, HeLa cells were infected with HSV-1 (at MOI (, 1) .The viral offspring produced at 3, 6, 9 and 12 hpi was titrated on elisa plate. HIPK2 induces an increase in the amount of HSV-1 produced relative to the conditions of production in cells transfected with a control siRNA (SiC) Figure 4: Quantification of the production of A / H3N2 viruses under over-expression conditions eGFP-HIPK2 by transfection of the plasmid encoding the eGFPHIPK2 fusion After transfection, the HeLa cells were infected with the A / H3N2 virus (at 0.1 MOI) The virus progeny produced at 16, 20 and 36 hpi was titrated with RT-qPCR The overexpression of eGFP-HIPK2 induces a decrease in the amount of A / H3N2 virus produced up to 20 hpi compared to the production conditions in cells transfected with a plasmid encoding the eGFP. Figure 5: Quantification of Ad5 production in overexpression condition of eGFP- HIPK2 by transfection of plasmid encoding the eGFP-HIPK2 fusion. After transfection, HeLa cells were infected with Ad5 (at MOI 0.1 and 1). The viral offspring produced at 48 and 56 hpi was titrated on 293 cells. Overexpression of eGFPHIPK2 induced a decrease in the amount of Ad5 at 48 and 56 hpi compared to the conditions of production in cells transfected with a coding plasmid. for the eGFP.

Figure 6: Quantification of the production of HSV-1 in overexpression condition of eGFP-HIPK2 by transfection of plasmid encoding the eGFP-HIPK2 fusion. After transfection, HeLa cells were infected with HSV-1 (at MOI 0.1, 0.5 and 1). Viral offspring produced at 6, 9 and 12 hpi were titrated on Elisa plate. Over-expression of eGFP-HIPK2 induces a decrease in the amount of HSV-1 at 6, 9 and 12 hpi compared to the production conditions in cells transfected with a plasmid encoding eGFP.

EXAMPLES

1. Modification of the subcellular localization pattern of endogenous HIPK2 during infection With influenza A / Moscow / 10/99 H3N2 virus A549 cells were infected with H3N2 virus at a MOI of 0.1 for 24 hours (hpi). After binding to 4% PFA, endogenous HIPK2 and viral nucleoprotein (NP) were immunodetected by anti-HIPK2 (rabbit polyclonal, Santa Cruz H55) and anti-NP (17C2 mouse monoclonal, FRE3011) antibodies and cell nuclei marked by the DAPI. The cells were observed by confocal microscopy. The subcellular localization pattern of endogenous HIPK2 is completely remodeled by infection at 30 hpi: the HIPH2 accumulation domains (visible in the nucleus of uninfected cells, m) have disappeared; HIPK2 labeling is diffuse in the nuclear space. Identical results were obtained in HeLa cells. Similar results were obtained with cells infected with HSV-1 or adenovirus type 5. In A549 cells infected with Adenovirus type 5 (Ad5). The localization pattern of endogenous HIPK2 is modified relative to uninfected cells. HIPK2 accumulates strongly in nuclear domains peripheral to the central nuclear zone of accumulation of viral material (in the form of rosary). In cells infected with an HSV-1 virus. The subcellular localization pattern of endogenous HIPK2 is modified relative to uninfected cells. HIPK2 is a prime target for viruses, which systematically modify the subcellular localization pattern.

II. Infection with influenza A / H3N2 virus induced a strong increase in endogenous HIPK2 expression. HeLa cells were infected with virtus A / H3N2 at a MOI of 0.1 for 8, 24 and 30 hpi. Total cell extracts were made and deposited on SDS PAGE polyacrilamide gel for characterization. Western blots were made with antibodies to reveal the proteins HIPK2, NP (infection marker) and cellular actin (standard control for the amount of material). Similar results were obtained with cells infected with HSV-1 or Adenovirus type 5: endogenous expression of HIPK2 is induced by HSV-1 and Ad5 virus infections. The endogenous cellular expression of HIPK2 is induced during stress such as viral infections. Like PML, HIPK2 could be a major player in a new cell (innate) anti-viral response pathway.

III. Endogenous HIPK2 subexpression resulted in a significant increase in A / H3N2 virus production. HeLa cells were transfected with control siRNA (SiC) or specific for HIPK2-encoding mRNA (SiHIPK2). At 48 hours post-transfection, the cells were infected with the H3N2 virus at a MOI of 0.1 for 8, 24 and 32 hpi. An immunofluorescence assay at 32 hpi was performed and revealed a significant decrease in endogenous HIPK2 expression in HIPK2 siRNA transfected cells compared to control siRNA (SiC) transfected cells. Labeling of the viral nucleoprotein in the nuclei of infected cells indicates a significant increase in the number of cells infected in the context of extinguishing endogenous HIPK2 expression. Quantification of the produced viruses was performed in both populations of control siRNA (SiC) and endogenous HIPK2 (SiHIPK2) expressing cells, as well as in a non-transduced (T) cell population ( Figure 1). Viral titration was performed by RTqPCR in order to quantify the viral RNA contained in the virions produced in the culture supernatant (FIG. 1). Under the condition of extinction of expression of HIPK2 (siHIPK2), the production of influenza virus is optimized.

Similar results were obtained for the HSV-1 and Ad5 viruses, the production of which is increased under the expression extinction condition of endogenous HIPK2 (FIG. 2 and FIG. 3).

IV. Over-expression of HIPK2 is unfavorable to the replicative cycle of A / H3N2 virus. HeLa cells were transfected with a pcDNA3 plasmid encoding HIPK2 protein merged with eGFP or only eGFP. At 48 hours post-transfection, the cells were infected with the H3N2 virus at a MOI of 0.1 for 16, 20 and 36 hpi. Quantification of the produced viruses was performed in both cell populations transduced by the plasmids encoding eGFP-HIPK2 and eGFP (Figure 4). Viral titration was performed by RTqPCR in order to quantify the viral RNA contained in the virions produced in the culture supernatant. Under overexpression of HIPK2 (eGFP-HIPK2), influenza virus production was significantly decreased at 16 and 20 hpi, compared to that obtained from cells overexpressing eGFP alone or untransfected (Figure 4). .

REFERENCES

BIBLIOGRAPHIC REFERENCES Giraud S et al., J Virol. 2004, 78 (6): 2984-93 Lohr et al., Vaccine, 2009, 27: 4975-4982 Patent references WO 2003/076601 WO 2004/108754 WO 2008/129058 WO 2009/059411 WO 2009/004016 US 6,303,371 EP 1862537 US 2006/0188977 US 2004/0142450 US 2005/0084503

Claims (15)

REVENDICATIONS1. A method of in vitro production of virus by cell culture comprising the infection of cells with a virus and the replication of the virus in these cells characterized in that the cells are modified cells in which the HIPK2 gene is inactivated or in which the expression of the HIPK2 gene is inhibited. 2. Process for producing in vitro virus by cell culture according to the preceding claim wherein the modified cells have a deletion of the HIPK2 gene. 3. In vitro method of producing virus by cell culture according to one of the preceding claims wherein the modified cells express an interfering RNA specific for HIPK2. 15 4. A method of producing in vitro virus by cell culture according to one of the preceding claims wherein the virus is selected from adenoviruses, HSV viruses and viruses of the family of orthomyxoviridae. 5. In vitro method of producing virus by cell culture according to one of the preceding claims wherein the virus is selected from influenza viruses of type A, B and C. 6. In vitro method of producing virus by cell culture according to one of the preceding claims characterized in that the cells are selected from mammalian cells, avian cells or insect cells. 7. In vitro method of producing virus by cell culture according to one of the preceding claims characterized in that the cells are selected from CHO, MDCK, COS, CV1, Vero, BHK21, PERCE, HEK, HeLa, AGE1.CR cells. , AGE1.CR.pIX, EB66, EBx and HEp-2. 8. Process for producing viral products comprising the in vitro production of virus by cell culture according to one of the preceding claims. 9. Cells modified for the production of viruses by cell culture characterized in that the HIPK2 gene is inactivated or in that the expression of the HIPK2 gene is inhibited; and in that the cells are infected with a virus. 10. Cells modified for the production of viruses by cell culture, characterized in that the cells are chosen from CHO, MDCK, COS, CV1, Vero, BHK21, PERCE, HEK, HeLa, AGE1.CR, AGE1.CR.pIX cells. EB66, EBx and HEp-2; and that the HIPK2 gene is inactivated or that the expression of the HIPK2 gene is inhibited. 11. Cells modified for the production of virus by cell culture according to claim 10 characterized in that the cells are infected with a virus. 12. Cells modified for the production of virus by cell culture according to one of claims 9 or 11 characterized in that the viruses are selected from adenoviruses, HSV viruses and viruses of the family of orthomyxoviridae. 13. Cells modified for the production of virus by cell culture according to claim 12, characterized in that the viruses are chosen from influenza viruses of type A, B and C. 14. Cells modified for the production of virus by cell culture according to one of claims 9-13, characterized in that the HIPK2 gene is inhibited by the expression of an interfering RNA specific for HIPK2. 15. Cells modified for the production of virus by cell culture according to one of claims 9-13, characterized in that the HIPK2 gene is inactivated by its deletion.