MXPA00010892A - Viral production process - Google Patents

Abstract

The present invention is directed to a method of producing recombinant viral vectors at high titers incorporating a variety of important advancements over the art. The method of the present invention incorporates multiple features which provide enhanced production of viruses, particularly those viruses encoding exogenous transgenes. The specifically illustrated method describes a method for the high titer serum-free media production of recombinant replication defective adenoviruses containing an exogenous transgene. The invention provides methods of preparing microcarriers, methods for seeding bioreactors at high cell density, increasing the infectivity of the producer cells to the virus, methods to increase product yield through synchronization of the cell cycle of the producer cells, and methods to minimize the deleterious effects of exogenous transgenes. The invention further provides producer cells prepared by the process of the invention. The invention further provides viruses produced by the process.

Description

VIRAL PRODUCTION PROCEDURE BACKGROUND OF THE INVENTION A variety of in vivo gene therapy products currently under development are based on the delivery of a therapeutic transgene by means of recombinant viral vectors. A common vehicle for the delivery of transgenes is recombinant adenoviruses, typically those deficient for replication in any other cell than a specific packaging cell line. These packaging cell lines express certain adenoviral genes necessary for the replication of viruses that have been eliminated in the deficient virus. For the reproduction of adenoviruses that contain deletions in the E1 region, the cell line most commonly used is cell line 293. The production of replication adenoviruses deficient in 293 cells is difficult because it is difficult for the cell line to grow. For example, 293 cells require attachment to a substrate and appear to differentiate at high confluence. Another limitation is that deficient replication adenoviruses do not replicate as well as wild type viruses. Although the production of wild-type adenovirus-specific viruses in 293 cells is about 80,000-100,000 particles per cell, the E1-deficient replication adenoviruses typically produce only 100-2,000 particles per cell. Based on current determinations of dosage regimens and therapeutic market sizes, calculations have indicated that annual production of approximately 1018 particles will be necessary to meet the demand for some gene therapy products. Therefore, improvements in the production of recombinant adenoviruses are required at levels that will satisfy the anticipated market for adenoviral gene therapy products to make this technology commercially possible. The present invention describes a microcarrier based process for the production of viral vectors in anchor-dependent packaging cell lines, which allows for the cost-effective production of adenoviral gene therapy products sufficient to meet the projected demand in the market. The invention describes a scalable production process that produces more than 2x1015 viral particles in a 5 liter bioreactor. This procedure is fully scalable to achieve the 1018 particles projected per year with a bioreactor as small as 100 liters and 5-liter purification columns.

BRIEF DESCRIPTION OF THE INVENTION The present invention is directed to a method for producing recombinant viral vectors at high concentrations that incorporates a variety of important advances in the art. The method of the present invention incorporates several features that provide for the increased production of viruses, particularly those viruses that code for exogenous transgenes. The specifically illustrated method describes a method for the production of high concentration serum free media of replication defective recombinant adenoviruses that contain an exogenous transgene. The invention provides methods for preparing microcarriers, methods for planting bioreactors at high cell density, increasing the infectivity of the producer cells for the virus, methods for increasing the product yield through synchronization of the cell cycle of the producer cells, and methods to minimize the deleterious effects of exogenous transgenes. The invention further provides producer cells prepared by the process of the invention. The invention also provides viruses produced by the process.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a photographic representation of microcarriers coated with cells examined under a light microscope. Panel A illustrates confluent microvehicles having approximately 10 6 cells / ml or an average of approximately 23 cells per microcarrier. Panel B demonstrates the results of the super cell concentration on the microcarriers of about 10 7 cells / ml or an average of about 230 cells per microcarrier.

Figure 2 is a graphical representation of the production levels of viral particles of ACN53 produced by the procedure described in Examples 1-5 hereof. The vertical axis represents the total number of viral particles in the bioreactor. The horizontal axis represents the time after infection in hours. In this example, 5x10 cells / ml were infected with virus, which resulted in the production of approximately 12,800 ACN53 viral particles per cell. Figure 3 is a graphic representation of the production of ACN-Rb110 virus substantially in accordance with the teaching of examples 1-5 of the present. The vertical axis represents the total number of viral particles. The horizontal axis represents the time after infection in hours. These data demonstrate the production of approximately 39,000 viral particles ACN-Rb110 per cell.

DETAILED DESCRIPTION OF THE INVENTION All publications, patents and patent applications cited in this disclosure are hereby incorporated by reference in their entirety as if each publication, patent or individual patent application was specifically and individually indicated as incorporated by reference. The use of the singular in the specific terms also implies the use of the plural and vice versa. Headings are included simply for convenience and are not intended to be limiting of the scope of the description. The production of viruses by mammalian cell cultures depends on a variety of factors. As described below, a variety of techniques can be employed to improve the production of viruses in a certain producer cell, such as synchronization of the producer cells, increase of the infectivity of the producer cells and suppression of the effects of transgenes during cultivation. Although it is theoretically possible to produce large quantities of viral particles by expanding the production scale or by repeating low yield procedures, these factors combine to curb the commercial practicality of such approaches. Consequently, the overall efficiency of the process in a certain volume depends, in large part, on the concentration of cells that can be effectively maintained in a certain volume of medium. If a high concentration of viable producer cells could be achieved in a certain volume, combined with a high production of intracellular viruses, the overall efficiency of the process would be improved to make the process economical.

I. Obtaining a high density of cells in a microcarrier-based reactor The present invention provides a method for achieving a cell density greater than 5x106 producer cells / ml in a microcarrier-based bioreactor process for the production of a virus in a producer cell, said method comprises the steps of: a) preparing a culture of producer cells fixed to microcarriers where the ratio of producer cells to microcarriers is about 10 cells / microcarrier; b) seeding the bioreactor with a quantity of the microcarriers coated with producer cells prepared in step a) to a density of more than about 6 grams (based on the dry weight of the microcarrier) of the microcarriers coated with producer cells per liter of volume of medium in the bioreactor and c) culturing the producer cells in the bioreactor under perfusion conditions in media containing serum at a density of more than 100 cells / microcarrier.

Bioreactor The term "bioreactor" refers to a device for growing cells that contains a container in which the cell culture is maintained. The design of the bioreactor should ensure sterility and provide containment of the genetically engineered producer cell and virus. A variety of bioreactors are commercially available for the culture of anchor-dependent producer cells, and suspension cultures are well known to those skilled in the art and can readily be adapted to the practice of the present invention. The bioreactors are preferably equipped with a stirring system to keep the contents uniformly mixed and to facilitate oxygen transfer. Preferably, the bioreactor includes sensors that allow the monitoring and manipulation of as many process parameters (temperature, pH, dissolved oxygen) as possible so that these parameters can be maintained within optimal scales for cell growth. A bioreactor in the preferred embodiment of the invention contains an apparatus for oxygenating the media, which is separated from the bed of the microcarrier. A preferred bioreactor for use in the practice of the present invention is the CelliGen Plus® bioreactor equipped with the Cell-Lift® impeller for low shear and high oxygenation in commercially available microcarrier crops from New Brunswick Scientific Company, Inc., 44 Talmadge Road, Edison, New Jersey, USA, 08818-4005. Certain modifications, such as a cell sedimentation tube or a decantation column, can be used to facilitate culture manipulation (commercially available from New Brunswick Scientific).

Virus and viral vectors The terms "viruses" and "vectors" are used interchangeably herein. The term "particles" or "viral particles" refers to virions or envelopes within which the viral genome is packaged. The viruses that will be produced by the practice of the present invention include recombinantly modified, enveloped and non-enveloped DNA and RNA viruses, preferably selected from the families Baculoviridiae, Parvoviridiae, Picomoviridiae, Herpesviridiae, Poxviridiae, Adenoviridiae or Picornnaviridiae. Viruses can be natural viruses or their viral genomes can be modified by recombinant DNA techniques to include the expression of exogenous transgenes and can be manipulated to be replication-deficient, replication-conditioned or replication competent. Chimeric vectors that exploit useful elements of each of the properties of the source vector can also be produced by the methods described herein (see, for example, Feng, et al (1997) Nature Biotechnology 15: 866-870). Also, minimal vector systems can be produced in accordance with the practice of the present invention in which the viral base structure contains only the sequences necessary for viral vector packaging and can optionally include a transgenic expression cassette. The present invention is particularly useful in the preparation of viruses that are derived from the adenoviral, viral adeno-associated and retroviral genomes. In the most preferred embodiment of the present invention, the vectors that will be produced are incompetent replication vectors derived from the human adenovirus genome. In the preferred embodiment of the invention, the vectors that will be produced are adenoviral vectors of deficient replication or conditioned replication. In the embodiment of the invention that is most preferred and exemplified herein, the vectors that will be produced are deficient replication-defective adenoviral (E1 defective E1) vectors encoding an expression cassette for the exogenous tumor suppressor gene in a cell infected by the vector. Viral vectors of conditional replication are used to achieve selective expression in particular cell types while avoiding a broad-spectrum unpleasant infection. Examples of conditioned replication vectors are described in Bischoff, et al. (1996) Science 274: 373-376; Pennisi, E. (1996) Science 274: 342-343; Russell, S.J. (1994) Eur. J. of Cancer 30A (8): 1165-1171. In addition, the viral genome can be modified to include inducible promoters that achieve replication or expression of the transgene only under certain conditions. Examples of inducible promoters are known in the scientific literature (see, for example, Yoshida and Hamada (1997) Biochem. Biophys., Res. Comm. 230: 426-430; lida, et al. (1996) J. Virol. 70 (9): 6054-6059; Hwang, er al. (1997) J. Virol 71 (9): 7128-7131; Lee, ef al. (1997) Mol. Cell. Biol. 17 (9): 5067-5105; and Dreher, et al. (1997) J. Biol. Chem 272 (46); 29364-29371. The transgene may also be under the control of a tissue-specific promoter region that permits expression of the transgene only in particular cell types. In some cases it may be valuable to use viruses that carry out the expression of the transgene in a particular cell type. Certain vectors exhibit a natural tropism for certain types of tissue. For example, vectors derived from the genus Herpesviridiae have been shown to have preferential infection of neuronal cells. Examples of recombinantly modified Herpesviridiae vectors are described in the patent of E.U.A. No. 5,328,688, issued July 12, 1994. Specificity of cell type or selection of cell type can also be achieved in vectors derived from viruses having characteristically broad infectivities by modification of viral envelope proteins. For example, cell selection has been achieved with adenovirus vectors by selectively modifying the protrusion and fiber coding sequences of the viral genome to achieve the expression of modified boss and fiber domains that have specific interaction with unique cell surface receptors. Examples of such modifications are described in Wickham, ef al. (1997) J. Virol 71 (11): 8221-8229 (incorporation of RGD peptides into adenoviral fiber proteins); Amberg, ef al. (1997) Virology 227: 239-244 (modification of adenoviral fiber genes to achieve tropism in the eye and genital tract); Harris and Lemoine (1996) TIG 12 (10): 400-405; Stevenson, ef al. (1997) J. Virol. 71 (6): 4782-4790; Michael, et al. (1995) Gene Therapy 2: 660-668 (incorporation of gastrin-releasing peptide fragment into adenovirus fiber protein) and Ohno, et al. (1997) Nature Biotechnology 15: 763-767 (incorporation of the protein A-IgG binding domain in Sindbis virus). Other methods of cell-specific selection have been achieved by conjugation of antibodies or antibody fragments to the envelope proteins (see, for example, Michael, et al. (1993) J. Biol. Chem 268: 6866-6869, Watkins. , Ef al. (1997) Gene Therapy 4: 1004-1012; Douglas, ef al. (1996) Nature Biotechnology 14: 1574-1578.

Alternatively, particular portions can be conjugated to the viral surface to achieve selection (see, for example, Nilson, et al. (1996) Gene Therapy 3: 280-286 (conjugation of EGF to retroviral proteins). Recombinant can be produced according to the practice of the present invention In the preferred embodiment of the invention, the virus that will be produced is derived from the genus Adenoviridiae.The viruses that are particularly preferred are derived from human adenovirus type 2 or type 5. Said viruses are preferably of poor replication by modifications or deletions in the E1a and / or E1b coding regions Other modifications to the viral genome are preferred to achieve particular expression characteristics or to allow repeated administration or decrease the immune response. recombinant adenoviruses that have complete or partial deletions of the coding region n E4, which optionally retain E4orf6 and E4orf6 / 7. The E3 coding sequence can be deleted but is preferably preserved. In particular, it is preferred that the operator region of the E3 promoter be modified to increase the expression of E3 to achieve a more favorable immunological profile for the therapeutic vectors. More preferred are human type 5 adenoviral vectors containing a DNA sequence encoding p53 under control of the cytomegalovirus promoter region and the tripartite leader sequence having E3 under control of the CMV promoter and the deletion of the E4 coding while retaining E4orf6 and Erorf6 / 7. In the embodiment of the invention that is most preferred and exemplified herein, the vector is ACN53, as described in Wills, ef al. (1994) Human Gene Therapy 5: 1079-1088.

Producer cells The term "producer cell" is used herein to describe a line of anchor-dependent viral packaging cells. Anchorage-dependent cells, or cultures derived from them, are those that will grow, survive or maintain their function optimally when fixed to a surface such as glass or plastic. The use of this term does not imply that the cells are normal or that they are neoplastic or not. Depending on the nature of the virus that will be propagated, the genome of the cell line can be modified to complement deletions in the viral genome used as the vector. When the vector is of competent replication, any of the anchor-dependent cell lines commonly used for culturing mammalian cells such as viral packaging cell lines can be used. Examples of such anchor-dependent cell lines commonly used as viral vector packaging cell lines are HeLa or 293 cells (Graham and Smiley (1977) J. Gen. Virol. 36: 59-72), and PERC cells. 6 (such as those described in the publication WO / 97/00326, application Serial No. PCT / NL96 / 00244).

In some applications, particularly when the virus is to be used for gene therapy applications, it is preferable that the vector be replication-deficient (or replication-defective) to prevent uncontrolled proliferation of the virus in the individual to be treated. In such cases, mammalian cell lines that have been manipulated are selected, either by modifying the genome of the producer cell to encode essential viral functions or by coinfection of the producer cell with an auxiliary virus, to express proteins that complement the effect of the deleted sequences of the viral genome. For example, when the viral vector to be produced is the HIV-1 vector or a recombinantly modified derivative thereof, the HIV-1 packaging cell line, PSI422, can be used, as described in Corbeau, et al. (1996) PNAS (USA) 93 (24): 14070-14075. Similarly, when the viral vector that will be produced is a retrovirus, the line of retroviral packaging cells derived from 293 human (293GPG) capable of producing high concentrations of retroviral particles, as described in Ory, et al. (1996) PNAS (USA) 93 (21): 11400-11406. In the production of minimal vector systems, the producer cell is manipulated (either by modification of the viral genome or by the use of viruses or auxiliary cosmids) to complement the functions of the parent virus making replication and packaging in virions possible. the line of producing cells.

In the case where the virus to be produced is a recombinant adenovirus made of poor replication by deleting the E1a and / or E1b functions, the 293 cell line is particularly preferred because of its ability to complement the E1a and E1b function adenoviral However, 293 cells can also be used for the expression of competent replication or conditioned replication adenoviruses. Examples of other cell lines that can be used for the production of E1 defective adenoviruses are PERC.6 cells (available from IntroGene, bv, PO Box 2048, Leiden, The Netherlands), which code for E1 in trans, and has shown that they possess excellent fixation to a microcarrier surface.

Transgenes Recombinant vectors that will be produced by the methods of the present invention may optionally contain a transgenic expression cassette. The term expression cassette is used herein to define a nucleotide sequence (DNA or RNA) containing regulatory elements and a transgene coding sequence to carry out the expression of the transgene in the target cell. Regulatory elements include promoters, enhancers, transcription terminators, polyadenylation sites, etc. The term "transgene" encompasses not only the sequence encoding wild-type proteins and allelic variations, but also sequences of homologous proteins from other organisms, as well as any mutations or truncations thereof that display essentially the same function as the polynucleotide or sequence of proteins. wild type proteins. Examples of transgenes that can be included in such vectors include tumor suppressor genes, cyclin dependent kinase inhibitors, cytotoxic genes, cytostatic genes, proapoptotic genes, prodrug activation genes, tumor specific antigens or antisense sequences. The term tumor suppressor genes (TSGs) refers to genes that when expressed in a target cell, are capable of suppressing the neoplastic phenotype. Examples of tumor-specific antigens include MART1 and gp100 (Zhai, et al (1997) J. immunotherapy 20: 15-25). Examples of tumor suppressor genes include the Rb gene of retinoblastoma and its variants Rb110 and Rb56, the MMAC-1 gene, the p53 gene, the DCC gene, the NF-1 gene and the erbA and erbB genes, p33 and p73. The term cyclin-dependent kinase inhibitors includes the genes p27kip,? 57kip2, p15ink4b, p18ink4c, p19ink4d, p16ink4a and p21sdi-1. The term "cytotoxic genes" refers to genes that are designed to have a toxic effect on the target cell, either alone or in conjunction with exogenous chemical agents (e.g., prodrug activation genes). Examples of such cytotoxic genes include DNA sequences encoding the cytotoxic domains of ricin, diphtheria, or Pseudomonas exotoxin, as well as the E311.6 gene of adenovirus, E1a. of adenovirus. Examples of prodrug activation genes include the thymidine kinase and cytokine deaminase genes. Pro-apoptotic genes include the p53 and p53 genes (eg, bax, bid, caspases, cytochrome c, etc.) and adenovirus E4orf4. Examples of other therapeutic transgenes that can be included in the vectors that will be produced by the practice of the present invention include interferons (alpha, beta, gamma and consensus), interferon a2b E2F-Rb fusion proteins, interleukins (eg, IL) -2, IL-4, IL-10), dopamine, serotonin, GABA, ACTH and NGF.

Microcarriers Most animal cells used in virus production are anchorage dependent and require attachment to a surface for optimal growth. In 1967, Van Wezel described the use of small particles (0.2 mm), microcarriers, for the growth of anchor-dependent cells. These microcarriers are suspended in the culture medium by gentle agitation so that a homogeneous environment is obtained. Since the cells are located on the surface, are subjected to mechanical stress and precautions must be taken to avoid separation of the cells from the surface by shear stress during cultivation. Macroporous spheres in which the anchorage dependent cells have the possibility of using the inner surface may also be employed to reduce the possibility that the shear forces may degrade the cells to be cultured. However, said microcarriers limit the surface area available for viral infection, so the parameters of viral infection must be adjusted. The microcarriers have been manufactured from different synthetic materials, including dextran, polyacrylamide and polystyrene. The attachment of cells to these charged microcarriers is mediated by ionic attractions. Many types of cells have a cell surface protein, fibronectin, which has a biospecific binding to gelatin, facilitating the use of microcarriers coated with gelatin. An advantage of the use of gelatin is its susceptibility to degradation with proteolytic enzymes, which allows the cells to be released from microvehicles with almost 100% viability by dissolving the gelatin matrix with trypsin. In the preferred practice of the invention, the microcarriers are Cytodex® microcarriers commercially available from Pharmacia Biotech AB, Uppsala, Sweden. The Cytodex® microcarriers are based on interlaced dextran spheres. The spheres are transparent, spherical and hydrated, and are replaced with positively charged groups. The microcarriers have an average diameter of approximately 200 μm and a density of 1.03 g / cm3. Cytodex® has been derived to form three types: Cytodex® 3 is coated with collagen. In the preferred embodiment of the invention as exemplified herein, the microcarrier is Cytodex® 1. Cytopore® microcarriers are based on an interlaced cotton cellulose matrix. They are hydrophilic DEAE exchangers with an average diameter of 230 μm and a density of 1.03 g / cm3. The microcarriers are transparent and are easily transported through pipes. The macroporous Cytopore® protects the cells from shear forces generated by the agitator or the aerated or centrifugal filter. The matrix has an average pore size of 30 μm, which makes it possible for the cells to enter the interior of the microcarrier. Inside the microcarriers, the cells are protected from the cutting forces of the agitator, centrifugal filter or bubbles created by the bubbling. The microporosity of the Cytopore® facilitates the supply of nutrients to all sides of the cells.

Fixation of cells to the surface of microcarriers A culture of producer cells fixed to microcarriers is produced when the microcarriers make contact with the producer cells in a medium containing serum and are subjected to conditions that allow growth. The cells grow in the presence of the microcarriers and produce new daughter cells, which are transferred to the exposed surface of the microcarrier by shaking the culture. After concluding the described procedure, the cells are concentrated at a high density of more than 100 cells per microcarrier. This high concentration of cells on the microcarrier facilitates the production of high level virus. To achieve a high density of cells and facilitate the attachment of daughter cells to microcarriers a variety of methods can be employed. The conditions should be designed to ensure efficient transfer of the daughter cells to the surface of the microcarrier without discharging the progenitor cell from the surfaces of the microcarrier. In the situation where there is a low initial quantity of producer cells, the cells can be micro-crushed from the microcarriers by passing the coated microcarriers through a hole at a low pressure (approximately 1,406 kg / cm 2). The detached cells can then be used to uniformly seed a larger number of microcarriers. Alternatively, the bioreactor can also be sown directly with a large number of cells. The microcarriers can also be introduced into a flask containing medium. The microcarriers will sink to the bottom of the flask and cells are introduced into the flask which also sink and attach to the surface of the microcarrier. A slight agitation facilitates the fixing process. The concentration of cells on the surface of the microcarriers can easily be determined by light microscopy. As the cell culture expands, it is necessary to monitor and control the culture parameters such as the concentration of dissolved oxygen, pH, temperature and agitation. The pH must be monitored throughout the cell growth process to ensure optimal conditions for cell replication. As the cells grow, metabolites are released into the medium, a process that can change the pH of the medium. Therefore, the pH of the medium must be carefully monitored and adjusted by the addition of bases or acids to maintain a relatively constant pH. The precise pH that facilitates optimal growth will vary somewhat with the particular cell line, but is generally on the physiological pH scale. For 293 cells it is preferred that the pH in the cell culture be maintained on the scale of about 6 to about 9, most preferably about 7 to about 8, more preferably about 7.2 to about 7.5, still more preferably about 7.2 . Temperature is another physiological parameter that must be monitored and controlled. The temperature in the cell culture should be stabilized at the optimum growth temperature of the cell line to achieve a high cell density. Mammalian cells have an optical growth temperature. If they are grown at a temperature below optimal, cell growth occurs slowly. On the other hand, if the growth temperature is too high, the death of the cells could occur. In the preferred embodiment of the invention, wherein the producer cell line is cell line 293, the temperature should be kept below about 40 ° C, most preferably on the scale of about 30 ° C to about 38 ° C, more preferably around 37 ° C.

Sowing The term "seed" as used herein, describes the introduction of producer cells to the volume of the microcarrier bed. The microcarriers coated as those prepared in example 1 below, they are then introduced into a bioreactor. The bioreactor is "seeded" with a quantity of the coated microcarriers of approximately 6 grams (based on the dry weight of the microcarrier) of microcarriers coated per liter of bioreactor volume. This is substantially more than the volume recommended for planting microcarriers in a certain volume. It is common in the art that the coated microcarriers should not occupy a volume of more than about 5% of the container / medium volume, i.e. about 2 grams (based on the dry weight of the microcarrier) of microcarriers coated per liter of volume of medium / container. In the embodiment of the present invention, about 10 grams (based on the dry weight of the microcarrier) of volume of pelleted microcarriers, comprising about 20% of the volume of the container, are used. This high concentration of microcarriers contributes to a high density of recombinant virus cells in the final culture, and increases viral yield. To achieve high yields in the practice of the present invention, the concentration of coated microcarriers should be from about 6 to 25 grams (based on the dry weight of the microcarrier) per liter of reaction volume, preferably 6 to 15 grams and most preferably about 10 grams (based on the dry weight of the microcarrier) per liter of reaction volume.

Growth of cells in media containing serum at a high density of cells The cells are then cultured in the bioreactor under perfusion conditions. The cultivation of mammalian cells under perfusion conditions is well known in the art. However, it should be noted that certain parameters must be optimized to achieve maximum cell growth. For example, it is necessary to monitor the oxygen content throughout the crop. Since oxygen is sparingly soluble in water (8.4 mg / L at 25 ° C), the growing culture must be supplied continuously in the form of sterilized air or pure oxygen. The concentration of dissolved oxygen should be maintained in the range of about 5% to about 200%, preferably about 50% to about 120%, most preferably about 100%. The concentration of dissolved oxygen is defined as the point at which 100% of dissolved oxygen represents the concentration of oxygen dissolved in the medium in equilibrium with air. The oxygen concentration should be measured by conventional means such as by means of dissolved oxygen monitoring probes commercially available from Instech Laboratories, Inc., 5209 Militia Hill Road, PIymouth Meeting PA 194662-1216 or Lazar Research Laboratories, Los Angeles CA. Proper agitation of the bioreactor culture is essential to ensure an adequate supply of nutrients and to prevent the accumulation of toxic metabolites within the bioreactor. The agitation of the medium also affects the rate of oxygen transfer. Excessive agitation can cause mechanical damage to mammalian cells. Foaming must be avoided, since the bubbling associated with the foaming process can generate sufficient shear forces within the culture to cause the displacement of the cells from the surface of the microcarrier or the lysis of the cells. Therefore, a balance must be maintained between the need to provide adequate mixing and the need to avoid damage to cells. In the preferred embodiment of the invention as exemplified herein using a 5 liters CelliGen Plus bioreactor, agitation of about 70 rpm is maintained. To minimize foam formation, the medium is separated from the cells and oxygenated before reintroduction to the cells by using a bubbling attachment to the bioreactor.

II. Production of viral particles in the packaging cell line The method of the invention further provides a method for producing a population of producer cells containing a high concentration of viral particles in a bioreactor based on microcarriers in serum free media, said method it comprises the process described above and further comprises the steps of: d) removing the medium containing serum; e) synchronizing the producer cells in the G1 phase; f) infecting the producer cells with a virus; g) culture cells under conditions that allow viral replication until a maximum point is reached.

To prepare the bioreactor for the introduction of serum-free media, the agitation in the bioreactor must first be allowed to stop. The microcarrier spheres will then fall out of the suspension and settle to the bottom of the reactor volume, which allows the serum containing medium to be ned. The spheres are then washed with serum free medium to minimize the percentage of medium containing serum. Washing is achieved by returning the suspension of the microcarrier to its original volume with serum free medium and achieving homogeneous suspension of microcarriers by sufficient agitation. This medium is then ned as described above. The washing step can be repeated to achieve maximum removal of media containing serum. The serum-free medium is added to the cells in an amount sufficient to support cell culture during infection. The serum free medium is defined as the growth medium for animal cell cultures substantially free of animal derived sera. The serum free media is well known in the art (see, for example, Freshney, R.I., Culture of Mammalian Cells, 1983, pp. 76-77). Alan R. Liss Company, New York). The simple serum free media can be supplemented with additional factors sufficient to make cell growth possible. Alternatively, complete serum free media sufficient to support the growth of mammalian cells are commercially available from a variety of commercial suppliers. Examples of serum free media that are preferred include JRH Ex-Cell 525 serum free media (catalog number 61129-79P), CellGro serum free media (commercially available from Gibco-BRL Life Sciences, Gaitherburg Maryland, USA), media serum free HyClone (# A-11 11 -L) commercially available from HyClone.

Additives for media Serum-free media are often supplemented with one or more hormones such as insulin, transferrin, epidermal growth factor, hydrocortisone, etc. The serum free media can also be enriched with additional agents to facilitate cell growth and may depend on the cell line used in the virus to be produced. For example, the media can be enriched with TGF-beta or agents that upregulate endogenous transformation (TGF) -beta growth factor in the cell. Alternatively, agents that over-regulate or stabilize viral binding receptors, such as avß3 and avβd integrins, can be added to the culture to improve infection efficiency. In the most preferred embodiment of the invention as exemplified herein, serum free media are Biowhttaker # 12-604-F Dulbecco's Modified Eagle's Medium (DMEM) containing 1% CMF-1 (Applied Nutrient Sciences, Sorrento Valley , CA).

Deletion of harmful transgenes In the event that the recombinant virus uses a very strong promoter (for example, CMV) for the expression of the transgene, the production of recombinant proteins will start competing with the necessary resources for optimal viral replication. Accordingly, it may be advantageous to add elements to serum-free media that supercoagulate or inhibit the transgene promoter. For example, when the transgene promoter is the cytomegalovirus (CMV) early promoter, elements such as neuramidase or tunicamycin can be added to suppress the CMV promoter during culture. Second, it is known that particular therapeutic transgenes can have a negative effect on the producer cell. For example, it is known that tumor suppressor genes such as p53 induce apoptosis in normal cells with sufficient cell dosage. Consequently, viruses expressing said transgenes are particularly difficult to grow at high density. For example, a comparison of the yields of the embodiment of the present invention shown in Figures 2 and 3 demonstrates the significantly lower viral yield as a result of the expression of the p53 tumor suppressor gene. The present invention provides a method for minimizing the negative effect of a transgene toxin to the producer cell by adding an agent to the culture media in a concentration sufficient to inhibit the promoter that leads to the transgene. The agent that will be added will depend on the promoter used to drive the expression of the transgene, but it should not interfere materially with expression of viral genes essential for viral replication. For example, the cytomegalovirus major immediate early promoter is a promoter commonly used to constitutively drive transgenic expression. This promoter contains binding sites for the transcription factor NF-kB and requires the activated form of NF-kB for its activity. See, for example, Bellas, et al. (1995) J. Clinical Investigations 96: 2521 and Loser, et al. (1998) J. Vírology 72: 180-190. In the presence of compounds that inhibit the activation of NF-kB, such as N-acetyl-L-cysteine or pentoxifylline, the activity of the CMV promoter can be repressed to previous levels (Bailas, et al., Supra). This transient repression of the CMV promoter by adding said agents to the cultures during the production phase of recombinant viruses encoding cytotoxic / cytostatic transgenes driven by CMV promoter will improve the performance of said viruses. The effective concentrations of N-acetyl-L-cysteine and pentoxitilin are approximately 10 to 30 mM and 0.5 to 3.0 mM, respectively. Similarly, inhibitors of NF-kB activity are useful to prevent transgenic expression in situations where HIV-1-LTR is used to drive transgene expression (Mhashilkar, et al., J. Virology 71: 6486-6494). Examples of agents capable of suppressing the action of other promoters and their effective concentration scales are known in the art and can be substituted in the practice of the present invention.

In the case of the cytotoxic / cytostatic transgene under the control of an active promoter only in (or mainly) a particular type of cell, it is preferred to employ a producer cell line in which the cell-specific promoter (or tissue) is inactive . For example, when the promoter is active only in liver cells (see, for example, a-fetoprotein promoter (Huber et al., PNAS 88: 8039-8043) the producer cell line is preferably not derived from a cell line. In addition, in the case of inducible promoters such as those described above, it is preferred that the culture conditions (chemical composition, temperature, etc.) be maintained to avoid expression of the transgene from the inducible promoter. It is known to those skilled in the art that the above procedure for suppressing the effect of the transgene can be applied to any method for culturing eukaryotic cells, including but not limited to, micro-carrier-based cultures, suspension cultures, centrifugal culture and the so-called cell culture. of "rolling bottle".

Synchronization To achieve maximum performance, it is preferred that the cells be synchronized in the G1 phase before infection with the recombinant virus. By keeping the cells in the G1 phase, they are optimally prepared to be pushed into the S phase, where nucleic acid synthesis occurs mainly. By synchronizing the cells, peak intracellular viral production is achieved. In the absence of synchronization, the subpopulation of advanced producer cells in the cell cycle will undergo lysis of viral cells before the point is harvested optimally. These virions, which are then thrown into the supernatant of the bioreactor, will be lost after harvesting the cells. Similarly, cells that fall behind the G1 phase will not reach optimal viral concentrations at harvest time. Alternatively, another mechanism for the production of improved virus by synchronizing the producer cells is the stability of the virus or viral DNA not encapsidated after infection. For example, it has been shown that retroviruses have a half-life of 4-6 hours after infection and that higher rates of infection can be observed when cells are synchronized. Andreadis, ef al. (1997) J. Virol. 71: 7541-8: Andreadis, et al. (1998) Biotechnology and Bioengíneering 58: 272-281; Andreadis, et al. (1996) J. Theor. Biol. 182: 1-20. A variety of means can be employed to synchronize the cells in the G1 phase. Maintaining the producer cells in serum-free media will synchronize the cells in the G0 / G1 phase. Optimally, the cells are maintained in serum-free media for about one third of a cell cycle (approximately 6-8 hours) to completely synchronize the cells. Alternatively, agents that synchronize the cells can be added. Examples of such agents include TGF-beta which causes cell cycle arrest in the G1 / S interface. Similarly, phosphatidinositol 3-kinase inhibitors (e.g., wortmanin and LY294002 (Eli Lilly and Company)) will block cells in the G1 phase. Bacqueville, ef al. (1998) Biochem, Biophys, Res. Comm. 244: 630-636. In addition, proteasome inhibitors such as lactacystin and / or N-carbobenzoxy-L-leucyl-L-leucyl-L-norvalinal have been shown to induce cell cycle arrest in the G1 and G2 phases of the cell cycle. Mutomba, et al. (1997) Mol. Biochem. Parisitol 90: 491-504. Examples of other compounds that can be added to synchronize the cells include mimosine and aphidicolin (Oncogene (1997) 15 (22) 2749-2753), quercetin (Shen and Webber (1997) Oncol. Res. 9: 597-602), epirubicin ( Hedenfalk, ef al. (1997) Cytometry 29 (4): 321-327) and lovastatin (Molecular and Cellular Biology (1985) 6 (9): 1197-1213). It will be readily apparent to those skilled in the art that the above procedure for increasing yield by synchronizing the cells can be applied to any method for culturing eukaryotic cells, including but not limited to, microcarrier-based culture, suspension culture. , centrifugal culture and the so-called "bottle rolling" cell culture.

Line infection of virus-producing cells In cells that have been infected by multiple copies of a certain virus, the activities necessary for viral replication and virion packaging are cooperative. In this way, it is preferred that the conditions be adjusted so that there is a significant likelihood that the producer cells will be infected multiple times with the virus. An example of a condition that increases the production of virus in the producer cell is an increased concentration of virus in the infection phase. However, it is possible that the total number of viral infections per producer cell may be exceeded, resulting in toxic effects to the cell. Accordingly, it could be difficult to maintain the infections at the virus concentration on the scale of 106 to 1010, preferably about 109 virions per ml. Chemical agents can also be used to increase the infectivity of the producer cell line. For example, the present invention provides a method for increasing the infectivity of producer cell lines for viral responsiveness by the inclusion of a calpain inhibitor. Examples of calpain inhibitors useful in the practice of the present invention include calpain inhibitor 1 (also known as N-acetyl-leucyl-leucyl-norleucinal, commercially available from Boehringer Mannheim). It was observed that the calpain 1 inhibitor increased the infectivity of producer cell lines to recombinant adenoviruses. lll. Culture, harvest, lysis and purification During the phase in which viral replication is proceeding, the bioreactor is continuously fed with serum-free supplemented media. The oxygen concentration should be maintained at a level of about 50% to about 120% dissolved oxygen, preferably about 100% dissolved oxygen. To maximize the intracellular concentration of viral particles, the accumulation of virus particles within the cells should be monitored. In the preferred method, the viral concentration is determined by HPLC using a Resource Q® column as described in Example 7 hereof. When the level of viral particles begins to reach a plateau, the bioreactor stops and the cells are harvested. The invention further provides a method for producing intact viral particles, comprising steps (a) - (g) above and further comprising the steps of: h) harvesting the cells; i) lysing the producer cells; j) isolate the viral particles and k) purify the intact viral particles. When the concentration of viral particles is optimized as determined above, the complete contents of the bioreactor are removed and regulated at pH to maintain a pH of about 7.0 to about 8.5. At this point the cells can be frozen for storage at -70 ° C.

Lysis and purification When it is desired to isolate the viral particles from the producer cells, the cells are lysed, using a variety of means well known in the art. For example, mammalian cells can be used under low pressure conditions (differential pressure of 7.03-14.06 kg / cm2) or conventional freeze-thaw methods. Exogenous free DNA / RNA is removed by degradation with DNase / RNase. The viral particles are then purified by means known in the art. Chromatographic or centrifugation methods with differential density gradient can be used. In the preferred embodiment of the invention, the virus is purified by column chromatography in substantial accordance with the method of Huyghe et al. (1995) Human Gene Therapy 6: 1403-1416 as described in co-pending US patent application Serial No. 08 / 400,793 filed March 7, 1995.

EXAMPLES As will be apparent to those skilled in the art to which this invention pertains, it may be incorporated in forms other than those specifically described below. The particular embodiments of the invention described in the following examples are, therefore, considered as illustrative and not restrictive of the scope of the present invention.

EXAMPLE 1 Preparation of microcarriers Cytodex 1 microcarrier spheres were prepared by swelling and hydration in PBS. The PBS for this procedure was prepared by 1: 10 dilution of 10x phosphate buffered saline (PBS) with Milli-Q water (pH 7.5). 10 grams of Citodex 1 microcarrier spheres were added to a clean 500 ml bottle. Approximately 300-400 milliliters of PBS was added to the bottle and the contents were stirred until the microcarriers were hydrated and completely suspended (approximately 3 minutes). The microcarriers were allowed to settle for approximately 10 minutes. The PBS was removed by aspiration. The washing procedure was repeated twice. The microcarriers were resuspended in a final volume of 300 ml of PBS. The microcarriers were sterilized in an autoclave for 30 minutes at 250 ° C. The bottle containing the sterile microcarriers was transferred to a laminar flow vessel. EEI PBS was removed by aspiration and the spheres were washed once as above with sterile PBS. The microcarriers were then washed once in sterile DMEM containing 10% fetal bovine serum (FBS). The microcarriers were resuspended to a final volume of 300 ml with sterile DMEM containing 10% FBS.

EXAMPLE 2 Preparation of 293 GT cells ml of sterile DMEM containing 10% FBS was placed in a 15 ml conical tube. A flask of 293 GT cells containing approximately 5 × 10 6 cells was obtained by storage under liquid nitrogen and thawed in a 37 ° C water bath. The exterior of the bottle was washed in 70% isopropanol and opened to a laminar flow vessel. The contents of the bottle were transferred to the tube containing the medium. The tube was then centrifuged at 1,000 RPM in a Beckman TJ6 centrifuge for approximately 5 minutes. The supernatant was then removed by aspiration and the pellet resuspended in 10 ml of medium. The suspension was then transferred to a T-225 flask and incubated at 37 ° C for about 50 hours. The cells were allowed to grow at approximately 50-90% confluence determined by light microscopy. After reaching sufficient confluence, the medium was removed from the flasks and 10-30 ml of PBS were carefully pipetted along the top of the flasks. The flasks were then stretched, coating the cells with PBS. PBS was removed from the flasks and 10 ml of a 0.05% trypsin solution (0.05% trypsin, 0.53 mM EDTA commercially available from Gibco BRL under catalog number 25300-054) was added. The flask was gently rocked to ensure that the trypsin solution covered the monolayer. After about 45 seconds, each flask was shaken vigorously until the cells were completely detached from the flask. Immediately, 20 ml of complete medium was added to each flask. The contents of the flask were pooled by centrifugation (1, 000 RPM in a Beckman TJ6 centrifuge for approximately 5 minutes). The supernatant was decanted from a centrifuge tube, ground and the cells pooled using complete media. Cell count was determined by the use of a Reichert Bright-Line hemapitometer (Buffalo, NY). The culture was divided into four T-25 flasks (approximately 5x106 cells per flask). 10 ml of suspension were pipetted into each flask to be seeded. Complete medium was added to each of the flasks to a volume of 50 ml. The cells were incubated at 37 ° C in a humidified 7% carbon dioxide atmosphere. The cells were then expanded using the above procedure to 20 T-225 flasks. This procedure was repeated until a sufficient amount of cells was prepared to seed the microcarriers, approximately 6.5 x 108 cells.

EXAMPLE 3 Sowing the microcarriers Approximately 6.5 x 108 human 293 cells prepared substantially in accordance with Example 2 above were suspended in 5 liters of medium containing Biowhittaker # 12-604-F Dulbecco's Modified Eagle's Medium (DMEM) serum (commercially available from Biowhittaker, Inc., Walkersville MD) and 50 grams of microcarriers prepared substantially in accordance with Example 1 above. The above mixture was stirred well by swirling for a period of about 2 minutes. The entire mixture was then siphoned into the clean sterilized culture vessel of a 5-liter CelliGen Plus® bioreactor equipped with a Cell-Lift impeller (obtained from New Brunswick Scientific Company, Inc., 44 Talmadge Road, Edison, New Jersey, USA). 08818-4005). The cells were then cultured at a temperature of 37 ° C, maintaining a dissolved oxygen concentration of approximately 100%. The concentration of dissolved oxygen was monitored and maintained by using a probe for dissolved oxygen attached to a controller (part of the CellíGen Plus bioreactor) so that the level of dissolved oxygen was maintained at approximately 100% by bubbling with air, CO2, 02 or N2 as appropriate. The culture was maintained under atmospheric pressure and at a pH of about 7.2. The vessel was agitated at a rate that depended on the volume of the bioreactor vessel and may vary from about 15 to 200 rpm. For the 5 liter bioreactor used here, the stirring speed was maintained at about 70 rpm. The cells were cultured until the cell density reached the desired level (8-10 x 106 cells per milliliter or approximately 230 cells per microcarrier). The cell density can also be determined by the rate of oxygen consumption, which is proportional to the concentration of cells. After about 3 days of incubation, the reaction vessel was continuously fed with medium at the amount of about 5.0 liters per day per 1 x 106 cells. The consumed medium was removed by means of a decanting column. The cells were allowed to grow for a period of about 14 days. To determine the number of cells per milliliter, stirring was increased to 100 rpm and a 5 ml sample was quickly drawn using a sterile syringe. The contents of the syringe were then transferred to a 15 ml conical tube. When the microcarriers were pelleted, the supernatant was removed by aspiration and replaced with identical volume of 0.25% trypsin solution (0.25% trypsin, 1 mM EDTA, commercially available from GibcoBRL under catalog number 25200-056). The tube was capped and incubated in a 37 ° C water bath for approximately 5 minutes (or until the cells reached a hirsute appearance on the microcarriers). The tube was then vortexed for 20 seconds and as soon as the microcarriers began to settle (approximately 1 minute), a top sample was taken from the microcarrier layer for counting by the hematocytometer.

EXAMPLE 4 Media exchange Once the cells reached the desired density, the temperature controller, agitator and perfusion were turned off and the microcarriers were allowed to settle. Once the microcarriers sedimented, the medium was removed by siphoning. The reactor was filled with 5 liters of DMEM (without serum or additives, Biowhittaker). The stirring was ignited sufficiently to separate the microcarrier / cell cake (approximately 150 rpm) for about 3 minutes. Agitation was stopped and the coated microcarriers were allowed to settle. This procedure was repeated twice. The reactor was then filled with 5 liters of DMEM containing 1% CMF-1 (Applied Nutrient Sciences, Sorrento Valley CA). Stirring was resumed to separate the cake from cells and then adjusted to 70 RPM to maintain the suspension of the coated microcarriers. The temperature controller was turned on to maintain a temperature of 37 ° C.

EXAMPLE 5 Infection of packaging cell line 293 with recombinant adenovirus (ACN53) x 1012 particles of recombinant adenovirus were added (ACN53 described in Wills, ef al (1994) Human Gene Therapy) to the culture vessel prepared in example 4 above. The perfusion was resumed as in example 3 above. After approximately 20 hours of incubation, the reaction vessel was analyzed to verify the virus concentration. This was achieved by increasing the agitation at 100 rpm and rapidly extracting a 5 ml sample using a sterile syringe. The contents of the syringe were then transferred to a 15 ml conical tube. 0.5 ml of a HSM pH regulator (50 mM Hepes, 3% sucrose, 2 mM MgCl 2, 150 mM NaCl and 5% beta-cyclodextrin, pH 7.5) were added. The tube was then frozen in liquid nitrogen and thawed rapidly in a water bath at 10 ° C. This freeze / thaw process was repeated twice. The tubes were centrifuged at 3,000 RPM in a Beckman TJ6 centrifuge for approximately 5 minutes. A sample of supernatant was then extracted and determined by the Resouce Q® test as described by Shabram, et al. (1996-7) Human Gene Therapy. This procedure was repeated periodically (about every 2 hours) until it was determined that the viral concentration began to fall (ie, until the optimal viral concentration had been achieved). The results presented in data are shown in table 1 below and a graphic representation of the data is given in figure 2 of the attached drawings.

TABLE 1 Production of ACN53 in 293 CJT cells As can be seen from the data presented above, for ACN53 the optimal viral concentration is achieved approximately 50 hours after infection, although this will vary with individual constructs. The contents of the container were removed to freezer storage containers, mixed with 10-20% pH regulator HSM and frozen.

EXAMPLE 6 Harvesting and lysis of infected cells The complete contents containing the cells were used by a repeated freeze / thaw procedure. The contents were frozen in a freezer at -80 ° C and thawed in a water bath at room temperature. The virus was purified substantially according to the procedure of Huyghe et al. (1995) Human Gene Therapy 6: 1403-1416.

EXAMPLE 7 Production of ACN-Rb110 The procedure described in Examples 1-5 was repeated, except that a recombinant adenovirus expressing retinoblastoma protein p110 (ACNRbUO) was used to infect 293 cells. The construction of adenovirus ACNRB110 is described Smith, et al. (1997) Circulation 96: 1899-1905. The cell density at the time of infection was 1.0 x 10 7 cells / ml. The results of the infection culture are presented in Figure 3 of the accompanying drawings. In this example, a viral concentration of approximately 39,000 viral particles per cell was achieved.EXAMPLE 8 Improvement of the viral concentration of viral ACN-p53 using N-acetyl-L-cysteine A culture of producer cells 293 was prepared substantially in accordance with that indicated in the above examples 1-4. The cells are infected with ACN53 adenovirus as described in Example 5. However, the procedure of Example 5 is modified by the addition of N-acetyl-L-cysteine at a final concentration of about 10-30 mM in the medium of culture to inhibit the CMV promoter. Cells are grown and harvested substantially according to examples 5 and 6. The improved intracellular concentration of viral particles is the result of inhibition of expression of the p53 transgene.

Claims (30)

NOVELTY OF THE INVENTION CLAIMS

1. - A method to achieve a cell density of more than 5x106 producer cells / ml in a bioreactor based on microcarriers for the production of a virus in a producer cell, said method comprises the steps of: a) preparing a culture of fixed producer cells to microcarriers, wherein the ratio of producer cells to microcarriers is approximately 10 cells / microvehicle; b) seeding the bioreactor with a quantity of the microcarriers coated with producer cells prepared in step a) to a density of more than about 6 grams (based on the dry weight of the microcarrier) of the microcarriers coated with producer cells per liter of volume of medium in the bioreactor and c) culturing the producer cells in the bioreactor under perfusion conditions in media containing serum at a density of more than 100 cells / microcarrier.

2. The method according to claim 1, further characterized in that the producer cell is a cell 293.

3. The method according to claim 2, further characterized in that the virus is an adenovirus.

4. - The method according to claim 3, further characterized in that the virus is a defective replication adenovirus derived from the adenovirus type 5 genome.

5. The method according to claim 4, further characterized in that the defective replication adenovirus comprises in addition an expression cassette for an exogenous transgene.

6. The method according to claim 5, further characterized in that the exogenous transgene is selected from the group consisting of tumor suppressor genes, cytotoxic genes, cytostatic genes, proapoptotic genes or prodrug activating genes.

7. The method according to claim 6, further characterized in that the exogenous transgene is a tumor suppressor gene.

8. The method according to claim 7, further characterized in that the tumor suppressor gene is p53.

9. A method for producing a population of producer cells containing a high concentration of viral particles in a bioreactor based on microcarriers in serum free media, said method comprises the method according to claim 1, and further comprises the steps from: d) removing the medium containing serum; e) synchronizing the producer cells in the G1 phase; f) infecting the producer cells with a virus and g) culturing the cells under conditions that allow viral replication until a peak is reached.

10. - The method according to claim 9, further characterized in that the producer cell is a cell 293.

11. The method according to claim 10, further characterized in that the synchronization of cells is achieved by keeping the cells in a medium that it is not serum for more than about one third of a cell cycle.

12. The method according to claim 11, further characterized in that the virus is an adenovirus.

13. The method according to claim 12, further characterized in that the virus is a defective replication adenovirus derived from the type 5 adenovirus genome.

14. The method according to claim 13, further characterized by the replication adenovirus. defective also comprises an expression cassette for an exogenous transgene.

15. The method according to claim 14, further characterized in that the exogenous transgene is selected from the group consisting of tumor suppressor genes, cytotoxic genes, cytostatic genes, proapoptotic genes or prodrug activating genes.

16. A method for the production of viruses according to the method of claim 9, further comprising the steps of: h) harvesting the cells; i) lysing the producer cells; j) isolate the viral particles from the cell lysate and k) purify the intact viral particles.

17. - A purified virus produced by the method according to claim 16.

18. - A method for increasing the yield of a process for the production of virus in a producer cell by the addition of an agent that increases the infectivity of the producing cell for the virus.

19. The method according to claim 18, further characterized in that the virus is an adenovirus.

20. The method according to claim 19, further characterized in that the producer cell is a cell 293.

21. The method according to claim 20, further characterized in that the agent that increases the infectivity of the producer cell is inhibitory. of calpain 1.

22.- A method to increase the number of viral particles in a producer cell infected by a virus that contains a transgene toxic to the producer cell by the addition of an agent that suppresses the activity of the regulatory regions that drive the expression of the transgene.

23. The method according to claim 22, further characterized in that the transgene is a pro-apoptotic, cystatic or cytotoxic gene.

24. - The method according to claim 23, further characterized in that the virus is an adenovirus and the promoter is the cytomegalovirus early promoter.

25. The method according to claim 24, further characterized in that the agent that suppresses the activity of the cytomegalovirus early promoter is selected from the group consisting essentially of N-acetyl-L-cysteine and pentoxitiline.

26. The method according to claim 25, further characterized in that the concentration of N-acetyl-L-cysteine or pentoxitiline in the medium is approximately 10 to 30 mM and 0.5 to 3.0 mM, respectively. 27.- A method to increase the yield of a procedure for the production of virus in a producer cell by synchronizing the producer cells in the G0 / G1 phase before infection by the virus. 28. The method according to claim 27, further characterized in that the synchronization of the cells is achieved by maintaining the cells in a medium that is not serum for more than about one third of a cell cycle. 29. The method according to claim 28, further characterized in that the producer cell is a cell 293. The method according to claim 11, further characterized in that the virus is an adenovirus. * a m * '• *