Cell Lines

Abstract

The present invention relates to a process for producing a cell which constitutively expresses cytotoxic virus poly-peptides (e.g. VSV G or Gag-Pol). The invention also provides plasmids/vectors and kits for use in the production of the cells. Furthermore, the invention provides a process for producing retroviruses using the cells of the invention.

Claims

1. A process for producing a mammalian cell which constitutively expresses one or more cytotoxic virus polypeptides, the process comprising the steps: (i) introducing one or more nucleic acid molecules encoding (a) one or more cytotoxic virus polypeptides, and (b) one or more apoptosis inhibitors into a mammalian cell; and (ii) culturing the cell under conditions such that the one or more cytotoxic virus polypeptides and the one or more apoptosis inhibitors are constitutively expressed, wherein the expression of the one or more apoptosis inhibitors prevents apoptosis of the cell, and wherein the one or more nucleic acid molecules additionally comprises a selection gene which is located in the same primary transcript as the nucleic acid encoding the one or more cytotoxic virus polypeptides and is transcribed in the same mRNA molecule.

2. A process as claimed in claim 1, where the selection gene is inserted after an internal ribosome entry site (IRES) downstream of the stop codon of the nucleic acid encoding the one or more cytotoxic virus polypeptides.

3. A process as claimed in claim 1 or claim 2, wherein one or more of the cytotoxic virus polypeptides are selected from the group consisting of VSV G and Gag-Pol.

4. A process as claimed in any one of claims 1 to 3, wherein the mammalian cell is a human cell.

5. A process as claimed in any one of claims 1 to 4, wherein the mammalian cell is an immortalised cell, preferably a HEK 293 cell.

6. A process as claimed in any one of the preceding claims, wherein one or more of the apoptosis inhibitors is a polypeptide or RNA.

7. A process as claimed in any one of the preceding claims, wherein one or more of the apoptosis inhibitors is an inhibitor of the APAF-1, Caspase 9, BAK, BAX or BAD pathway.

8. A process as claimed in any one of the preceding claims, wherein the apoptosis inhibitors inhibit more than one of the APAF-1, Caspase 9, BAK, BAX or BAD pathways.

9. A process as claimed in any one of the preceding claims, wherein one or more of the apoptosis inhibitors is Celovirus GAM1, Adenovirus E4 Orf6, Adenovirus E1B 55K, Adenovirus FIB 19K, Myxomavirus M11L, Cytomegalovirus 1E1, Cytomegalovirus 1E2, Baculovirus p35, Baculovirus IAP-1, Herpesvirus US3, Herpesvirus Saimiri ORF16, Herpes Simplex 2 LAT ORF 1, Human XIAP, African Swine Fever ASFV-5-HL (LMW-5-HL/A179L), Kaposi's Sarcoma virus KSbcl2, Vaccinia virus SPI-2, Cowpoxvirus CrmA, Epstein Barr virus BHRF1, Epstein Barr virus EBNA-5, Epstein Barr virus BZLF-1, Papillomavirus E6, Human Aven, Human BCL2 or Human BCL-XL.

10. A process as claimed in claim 9, wherein one or more of the apoptosis inhibitors is AVEN or E1B-19K.

11. A process as claimed in any one of the preceding claims, wherein the one or more nucleic acid molecules encode two apoptosis inhibitor polypeptides.

12. A process as claimed in claim 11, wherein the two apoptosis inhibitors are AVEN and Adenovirus serotype 5 E1B-19K.

13. A process as claimed in claim 9, wherein the one or more nucleic acid molecules encode one, two or three apoptosis inhibitor polypeptides selected from the group consisting of Human XIAP, Kaposi's Sarcoma virus KSbcl2 and Epstein Barr virus BHRF1.

14. A process as claimed in claim 9, wherein the one or more nucleic acid molecules encode BHFR1 and also one or more apoptosis inhibitor polypeptides selected from the group consisting of BCL-XL, ASFV-5-HL and Vaccinina virus SPI-2.

15. A process as claimed in any one of the preceding claims, wherein the expression of the one or more cytotoxic virus polypeptides and/or the expression of the one or more apoptosis inhibitors is driven by one or more constitutive promoters.

16. A process as claimed in claim 15, wherein the one or more constitutive promoters are selected from the group consisting of the CMV, SV40, PGK (human or mouse), HSV TK, SFFV, Ubiquitin, Elongation Factor Alpha, CHEF-1, FerH, Grp78, RSV, Adenovirus EIA, CAG and CMV-Beta-Globin promoters, preferably from the group consisting of the RSV, CMV, SV40, PGK and ubiquitin promoters.

17. A process as claimed in claim 15, wherein the apoptosis inhibitors are human Aven and Adenovirus serotype 5 E1B-19K, and wherein the expression of human Aven and Adenovirus serotype 5 E1B-19K is driven by different promoters selected from RSV, CMV, SV40, PGK and ubiquitin promoters.

18. A mammalian cell which is obtained or obtainable by a process as claimed in any one of the preceding claims.

19. A cell line wherein the cells of the cell line constitutively express one or more cytotoxic virus polypeptides and one or more apoptosis inhibitors, wherein the expression of the apoptosis inhibitor(s) prevents apoptosis of the cells of the cell line, and wherein the cells of the cell line additionally express a selection gene product which is transcribed in the same mRNA molecule as the one or more cytotoxic virus polypeptides.

20. A cell line as claimed in claim 19, where the selection gene is inserted after an internal ribosome entry site downstream of the stop codon of the nucleic acid encoding the one or more cytotoxic virus polypeptides.

21. A cell line as claimed in claim 19 or claim 20, wherein one or more of the cytotoxic virus polypeptides are selected from the group consisting of VSV G and Gag-Pol.

22. A cell line as claimed in any one of claims 19 to 21, wherein one or more of the apoptosis inhibitors is Celovirus GAM1, Adenovirus E4 Orf6, Adenovirus E1B 55K, Adenovirus FIB 19K, Myxomavirus M11L, Cytomegalovirus 1E1, Cytomegalovirus 1E2, Baculovirus p35, Baculovirus IAP-1, Herpesvirus US3, Herpesvirus Saimairi ORF16, Herpes Simplex 2 LAT ORF 1, Human XIAP, African Swine Fever ASFV-5-HL (LMW-5-HL/A179L), Kaposi's Sarcoma virus KSbcl2, Vaccinia virus SPI-2, Cowpoxvirus CrmA, Epstein Barr virus BHRF1, Epstein Barr virus EBNA-5, Epstein Barr virus BZLF-1, Papillomavirus E6, Human Aven, Human BCL2 or Human BCL-XL, preferably AVEN or E1B-19K.

23. A cell line as claimed in claim 22, wherein the cells of the cell line constitutively express one, two or three apoptosis inhibitor polypeptides selected from the group consisting of Human XIAP, Kaposi's Sarcoma virus KSbcl2 and Epstein Barr virus BHRF1.

24. A cell line as claimed in claim 22, wherein the cells of the cell line constitutively express BHFR1 and also one or more apoptosis inhibitor polypeptides selected from the group consisting of BCL-XL, ASFV-5-HL and Vaccinia virus SPI-2.

25. A cell line as claimed in any one of claims 19 to 24, wherein the cell line is one which is capable of being passaged at least 5, more preferably at least 10 and most preferably at least 15.

26. A nucleic acid molecule encoding one or more cytotoxic virus polypeptides and one or more apoptosis inhibitors, wherein the nucleic acid molecule additionally comprises a selection gene which is located in the same primary transcript as the nucleic acid encoding the one or more cytotoxic virus polypeptides and is transcribed in the same mRNA molecule.

27. A nucleic acid molecule as claimed in claim 26, where the selection gene is inserted after an internal ribosome entry site downstream of the stop codon of the nucleic acid encoding the one or more cytotoxic virus polypeptides.

28. A nucleic acid molecule as claimed in claim 26 or claim 27, wherein one or more of the cytotoxic virus polypeptides are selected from the group consisting of VSV G and Gag-Pol.

29. A nucleic acid molecule as claimed in any one of claims 26 to 28, wherein one or more of the apoptosis inhibitors is Celovirus GAM1, Adenovirus E4 Orf6, Adenovirus E1B 55K, Adenovirus E1B 19K, Myxomavirus M11L, Cytomegalovirus 1E1, Cytomegalovirus 1E2, Baculovirus p35, Baculovirus IAP-1, Herpesvirus US3, Herpesvirus Saimairi ORF16, Herpes Simplex 2 LAT ORF 1, Human XIAP, African Swine Fever ASFV-5-HL (LMW-5-HL/A179L), Kaposi's Sarcoma virus KSbcl2, Vaccinia virus SPI-2, Cowpoxvirus CrmA, Epstein Barr virus BHRF1, Epstein Barr virus EBNA-5, Epstein Barr virus BZLF-1, Papillomavirus E6, Human Aven, Human BCL2 or Human BCL-XL, preferably AVEN or E1B-19K.

30. A nucleic acid molecule as claimed in any one of claims 26 to 29, wherein the nucleic acid molecule encodes one, two or three apoptosis inhibitor polypeptides selected from the group consisting of Human XIAP, Kaposi's Sarcoma virus KSbcl2 and Epstein Barr virus BHRF1.

31. A nucleic acid molecule as claimed in any one of claims 26 to 29, wherein the nucleic acid molecule encodes BHFR1 and also one or more apoptosis inhibitor polypeptides selected from the group consisting of BCL-XL, ASFV-5-HL and Vaccinina virus SPI-2.

32. A kit comprising: (i) a nucleic acid molecule encoding one or more cytotoxic virus polypeptides and a selection gene, wherein the selection gene is located in the same primary transcript as the nucleic acid molecule encoding the one or more cytotoxic virus polypeptides and is transcribed in the same mRNA molecule, and (ii) a nucleic acid molecule encoding one or more apoptosis inhibitors, for use in simultaneously, sequentially or separately introducing the nucleic acid molecules into a mammalian cell, in order to produce cells which constitutively express the one or more cytotoxic virus polypeptides and the one or more apoptosis inhibitors, and the selection gene product and wherein the expression of the apoptosis inhibitor(s) prevents apoptosis of the cells.

33. A process for producing retroviruses, the process comprising the steps: (a) introducing one or more nucleic acids encoding a retrovirus into a cell as defined in claim 18 or into a cell of a cell line as defined in any one of claims 19 to 25; (b) introducing one or more helper plasmids encoding one or more of Rev, Gag-Pol, or Tat polypeptides into the cell; (c) culturing the cell under conditions such that retroviruses are assembled and secreted by the cell; and (d) harvesting packaged retrovirus.

34. A process as claimed in claim 33, wherein the retrovirus is a lentivirus.

35. A process as claimed in claim 33 or claim 34, wherein two helper plasmids are used, wherein the first encodes Gag-Pol and the second encodes Rev.

36. A process for producing a recombinant polypeptide, the process comprising the steps: (a) producing retroviruses by a process as defined in any one of claims 33 to 35, wherein the retroviral genomes comprise a nucleic acid molecule encoding a desired recombinant polypeptide; (b) introducing the retroviruses into a host cell; (c) culturing the host cells under conditions such that the desired recombinant polypeptide is produced; and (d) harvesting the desired polypeptide.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0152] FIG. 1: Apoptotic efficiency of various gene/promoter combinations.

[0153] FIG. 2: Apoptotic efficiency of various gene/promoter combinations.

[0154] FIG. 3: Expression of VSV G in cell lines using antibodies against VSV G glycoprotein.

[0155] FIG. 4: Lentivirus production in transfected cell lines.

[0156] FIG. 4A: Cells infected with virus produced using standard four-plasmid lentivirus recovery.

[0157] FIG. 4B: Virus from stable cell line 1: produced by integration of genes encoding VSV and two genes known to inhibit apoptosis selected via puromycin resistance.

[0158] FIG. 4C: Virus from stable cell line 1: produced by integration of genes encoding VSV G and two genes known to inhibit apoptosis selected via puromycin resistance.

[0159] FIG. 5: Flow cytometry of cells infected with GFP expressing virus.

[0160] FIG. 5A: Cells infected with virus produced using standard four plasmid lentivirus recovery.

[0161] FIG. 5B: Virus from stable cell line 1, produced by integration of genes encoding VSV and two genes known to inhibit apoptosis selected via puromycin resistance.

[0162] FIG. 5C: Virus from stable cell line 1, produced by integration of genes encoding VSV G and two genes known to inhibit apoptosis selected via puromycin resistance.

[0163] FIG. 6: Stable cell lines expressing VSV G seeded at high density or confluence leads to the formation of large multi-nucleated syncytia.

[0164] FIG. 7: A plasmid schematic of a DNA molecule that could be used in the process of the invention by introduction into a mammalian cell. The diagram shows a DNA molecule encoding the VSV G glycoprotein and the apoptosis inhibitors human AVEN and Adenovirus E1B-19K.

[0165] FIG. 8: A plasmid schematic of a DNA molecule that could be used in the process of the invention by introduction into a mammalian cell. The diagram shows a DNA molecule encoding the VSV G glycoprotein and containing a Puromycin resistance gene downstream of an internal ribosome entry site (EMCV IRES) in addition to the apoptosis inhibitors human AVEN and Adenovirus E1B-19K.

[0166] FIG. 9: The effect of a range of genes (apoptosis inhibitors) on protein expression (luciferase) where each gene is under the control of the strong CMV promoter.

[0167] FIG. 10: The effect of a range of genes (apoptosis inhibitors) on protein expression (luciferase) where each gene is under the control of the weak SV40 promoter. Luciferase is driven by the CMV promoter.

[0168] FIG. 11: The effect of combining pairs of apoptosis inhibitor genes under the control of CMV and SV40 promoters, to give comparatively high and low levels of expression, respectively. Luciferase expression is driven by the CMV promoter.

[0169] FIG. 12: Human IgG antibody expression in suspension HEK-293 cells using a specific apoptosis inhibitor combination, namely KSbcl2 and BHRF1 both under regulatory control of the CMV promoter. Lanes 1: ladder. Lanes 2&3: competitor's plasmid at 12 g. Lanes 3&4: competitor's plasmid at 24 g. Lanes 15&16: plasmid containing a specific apoptosis inhibitor combination at 12 g. Lanes 17&18: plasmid containing a specific apoptosis inhibitor combination at 24 g. Apoptosis inhibitors allow the usage of higher DNA quantities without seeing the reduction in protein expression seen in Lanes 4&5.

[0170] FIG. 13: Yellow fever NS1 secreted protein expression measured via ELISA after 3 days from suspension HEK-293 cells. Sample A contains a standard CMV expression plasmid. Sample K contains a plasmid encoding KSbcl2 apoptosis inhibitor under regulatory control of the CMV promoter, shown here to significantly increase protein expression.

EXAMPLES

[0171] The present invention is further illustrated by the following Examples, in which parts and percentages are by weight and degrees are Celsius, unless otherwise stated. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

Example 1: Materials and Methods

DNA Engineering

[0172] All DNA constructs were synthesised by de novo synthesis where required. This process involved thermodynamically-balanced inside out oligo assembly to allow full length constructs to be produced from smaller oligos nucleotides (typically 4-50 nt in length). For the joining of DNA molecules, a combination of type II restriction endonuclease cloning and assembly PCR were used. The accuracy of each DNA construct was verified by restriction digestion and agarose electrophoresis and/or DNA sequencing using the Sanger method. All protocols are known to those in the art (e.g. Molecular Cloning: A Laboratory Manual (Fourth Edition), Michael R. Green, Joseph Sambrook, Cold Spring Harbor Laboratory Press).

Protocol for Creating Cell Lines

[0173] In order to establish a stable cell line expressing VSV G, HEK 293 cells were seeded in Dulbecco's Modified Eagle (DMEM) media (10% FCS, 1% penicillin/streptomycin) into T25 flasks (10 cm or 6 cm dishes may also be used) 24 hours prior to transfection so as to be at 80% confluent at time of transfection.

[0174] Cells were transfected using the PEI method. Briefly, the transfection mixture consisted of 15 g plasmid DNA in a 1:3 ratio with Branched PEI (25 KDa) respectively, added to two vials of 150p1 of DMEM (2% foetal calf serum (FCS)) Optimem media. Other media may be used, and preferably the media used would be Optimem to complex the DNA and would be free of both FCS and Penicillin and/or Streptomycin. The media/DNA mix and the media/PEI mix were combined and was incubated for 20 minutes at room temperature to allow complex formation. At the time of transfection, the pre-existing media in which the cells were seeded was removed by aspiration and changed for fresh DMEM media containing 10% FCS. Transfection mixtures were added drop-wise into the flasks and gently swirled to evenly distribute the transfection complexes in the media.

[0175] 24 hours post transfection, the media and transfection mixtures in each flask was removed by aspiration and replaced with DMEM (10% FCS, 1% penicillin/streptomycin) media containing puromycin which was added to the flasks at varying concentrations to determine the optimal antibiotic concentration required. For each DNA plasmid being tested, one flask was maintained at each of the following concentrations: 0 g/ml, 0.5 g/ml, 1.5 g/ml, 3 g/ml, 5 g/ml, 7.5 g/ml and 10 g/ml puromycin per ml of growth media.

[0176] Over the next 4 weeks, media in the flasks were changed every 3-4 days, maintaining the same concentrations of puromycin relevant to each flask, and the flasks were continuously evaluated for cell death and formation of cell foci/colonies. Formation of foci from single surviving cells was clearly observed in the flasks maintained at either 3 g/ml and 5 g/ml puromycin in flasks transfected with plasmids containing VSV G and the apoptosis inhibitors. After 4 weeks, two alternative approaches were taken. The contents of each of flask were either: [0177] 1. Gently trypsinized and passaged into larger T75 flasks, to make mixed population cell lines, maintaining the same puromycin concentration for each line. This was then followed by single cell isolation by limited dilution or FACS to identify single monogenic cell lines; or [0178] 2. Single colonies were encircled by a polycarbonate ring fixed temporarily to the flasks surface with sterile grease, followed by trypsinisation and colony isolation from within the ring and transfer to a 6-well plate containing the same media containing puromycin. The latter is the more preferable method of clonal cell line isolation.

[0179] After cells reached sufficient confluence, they were passaged with a 5-fold dilution; the remaining cells were analysed by flow cytometry. The cell lines were maintained from this point onwards at the same concentration of puromycin as originally selected in. Cell banks were created and stored at 170 C. using the cells remaining from each passage. In some instances, it may be possible to increase the VSV G expression by increasing the puromycin concentration in 1-2 g/ml increments, selecting for only the cells expressing the highest quantity of VSV G.

Protocol for Microscopy of Syncytia and GFP

[0180] Cell lines selected as described previously were seeded in 6 cm dishes so as to be 100% confluent after 24 hrs. The cell monolayer was observed for the formation of syncytia on a daily basis. After 5 days, cells were imaged using a Zeiss Axiovert Inverted microscope and imaged using a NIKON Coolpix camera with microscope adaptor. Where GFP images were required, samples were excited using ultraviolet excitation.

Protocol for FACS Analysis

[0181] A confluent T25 flask of cells from each cell line selected to express VSV G, as well as a flask of wild-type unmodified HEK 293 cells, were trypsinized and 5 ml of cell suspension were collected in 15 ml falcon tubes. These tubes were shaken at 37 C. for 1 hr to allow recovery of the VSV G glycoprotein on the cell surface after the trypsinisation process.

[0182] Each tube containing cells were then centrifuged at 1500 RPM for 5 minutes. Once pelleted, supernatants were removed by gentle aspiration and tubes were placed on ice. Cell pellets were re-suspended in PBS; 500p1 for the VSV G expressing cells and 750p1 for the HEK 293 cells. 250p1 amounts of the re-suspended cells were then aliquoted into 1.5 ml polypropylene tubes.

[0183] In order to stain cells to measure VSV G protein on the cell surface, solutions of primary antibody (anti-VSVG), secondary antibody (anti-mouse, FITC labelled) and isotype control (IgG) antibody were prepared at 4 g/ml in MACS buffer.

[0184] The cells in 1.5 ml polypropylene tubes were spun down at 5000 RPM in a benchtop centrifuge, the supernatant was removed, and the cell pellets were resuspended either in 200p1 MACS buffer+primary Ab or 200p1 MACS+isotype control Ab. Additionally, one tube of unmodified HEK 293 cells was suspended in 200p1 MACS alone, as an unstained cell control. The tubes were incubated at 12 C., shaking at 300 RPM, for 30 minutes.

[0185] After incubation, the samples were pelleted at 5000 RPM and washed twice in 250p1 PBS, pelleting between washes as described before. Samples were retained on ice as much as possible. Both the test and the control samples (apart from the unstained unmodified HEK 293 negative control cells) were then resuspended in MACS+ secondary Ab and incubated again at 12 C., shaking at 300 RPM, for 30 minutes. After the second incubation, the cells were pelleted as before and washed twice in PBS, and resuspended in a final volume of 250p1 PBS. Samples were analysed on a BD FACSCalibur using an Argon 488 Laser, gated for positivity against unstained HEK 293 cells.

[0186] In most experiments, stained cells were ready immediately after the staining protocol. However, when this was not possible (e.g. time periods over 4 hours between staining and analysis), cells were fixed by incubating in PBS 2% paraformaldehyde (PFA) for 15 minutes on ice, with gentle swirling to mix. The cells were then spun down and washed twice in PBS as before, resuspended in PBS, and stored at 4 C. Results from both methods consistently demonstrated high levels of VSV G on the surface of analysed VSV G cell lines.

Example 2: Apoptosis Inhibiting Genes

[0187] Gene expression was measured as a function of cell survival from apoptosis. Different genes inhibiting apoptosis were found to work with different efficiencies depending on their level of expression. For example, FIG. 1 shows that Gene 3 shows much improved activity under the promoter showing the highest expression level (CMV), whereas the weakest promoter (SV40) produces only a modest protection against apoptosis. However, Gene 2 shows the converse of this with the promoters showing the lowest expression level (SV40 and PGK) yielding the greatest protection against apoptosis. (In FIGS. 1-2, Gene 1 is Bcl-XL, Gene 2 is E1B-19K and Gene 3 is AVEN.)

[0188] FIG. 2 demonstrates that, when some inhibitors of apoptosis are combined, they can demonstrate improved resistance to apoptosis. In this assay, there is a strong trend towards cells expressing Gene 3 (AVEN) and Gene 2 (E1B-19K) showing improved resistance to apoptosis in comparison to either gene alone.

Example 3: Expression of VSV G in Cell Lines

[0189] FIG. 3 shows 4 individual cell lines (HEK 293 cells and stable cell lines expressing VSV G) which were stained using an antibody against the VSV G glycoprotein. In all cell lines produced to express VSV G, >98% of cells were highly positive for VSV G. HEK 293 cells showed no staining and an isotype control confirmed that the signal was specific to VSV G.

Example 4: Expression of Recombinant Lentiviruses in Transfected Cell Lines

[0190] HEK 293 cells were infected with a lentivirus expressing GFP that had been produced by either standard 4 plasmid transfection into HEK 293 cells (includes the VSV G plasmid) and compared to lentivirus produced in cell lines modified to constitutively express VSV G via 3 plasmid transfection (excludes the VSV G plasmid). FIG. 4 shows that, in all cases, more virus was found to be produced from cell lines which were constitutively expressing VSV G.

Example 5: Flow Cytometry of Cells Infected with GFP Expressing Virus

[0191] Cells infected with GFP-expressing virus produced by either standard 4-plasmid transfection into HEK 293 cells (includes the VSV G plasmid) or transfection of stable cell lines modified to express VSV G using a 3-plasmid transfection (excludes the VSV G plasmid) were analysed by flow cytometry. The results (FIG. 5) show that significantly more virus was produced from the stable VSV G cell lines in comparison to the industry-standard (i.e. 4-plasmid) method.

Example 6: Preliminary Screening for Lentivirus Production

[0192] VSV G expressing cell lines and wild-type HEK 293 cells were seeded at equal numbers in DMEM media (10% FCS, 1% penicillin/streptomycin) into three 6-well plates 24 hours prior to transfection so as to be at 80% confluence at the time of transfection 24 hours later. On each 6 well plate, 3 wells were seeded with wild-type HEK 293 cells, and each of the remaining 3 wells was seeded with cells from one of three selected VSV G expressing cell lines.

[0193] At time of transfection, DNA/PEI complexes were made up as follows:

[0194] For the VSV G expressing cells transfections, the following amounts of DNA/well were added to 395p1 of optimum per well in a master solution: [0195] 0.79 g Lentivirus GFP vector [0196] 0.79 g Rev plasmid [0197] 1.9 g Gag/pot plasmid

[0198] An amount of branched PEI equivalent to 3 the total weight of DNA was added to another 395p1 of optimum per well in a master solution for all wells.

[0199] Both the PEI and DNA solutions were filtered through a 0.2 micrometer sterile filter, and then the PEI solution was added dropwise to the DNA solution.

[0200] A solution which was identical in composition but also including 0.2 micrograms of VSV G plasmid was used for the transfection of the standard HEK 293 cell lines to allow comparison to standard lentivirus production systems.

[0201] Additional cells of HEK 293 cells were also seeded to act as negative controls and others seeded to generate a GFP positive control. For this control, a transfection complex identical to that described above using 7.4 g of a CMV-GFP plasmid vector only was used.

[0202] All transfection complexes were incubated for 20 minutes at room temperature in which time the seeded cells were washed with optimum medium, then 835p1 of each transfection complex was added dropwise to the respective wells. Cells were left to incubate at 37 C., 5% CO.sub.2 overnight and in the morning approximately 16-18 hours after transfection, the media was changed for DMEM (10% FCS, 1% penicillin/streptomycin). Supernatant from each well was then harvested at 48 hours post transfection and replaced with fresh media and harvested again at 72 hours after transfection. Supernatants were stored at 20 C.

[0203] In order to analyse the level of virus produced from each cell line, unmodified HEK 293 cells were seeded in DMEM (10% FCS, 1% penicillin/streptomycin) to a density of 90% in a 48 well plate 24 hours prior to infection. Two dilutions of harvested supernatant from each time point were used to infect the cells in triplicate wells. Dilutions were 2/5 and 4/25 into DMEM (10% FCS, 1% penicillin/streptomycin). Cells were infected by removing the overnight media, and adding 500p1 of diluted supernatant.

[0204] Cells were incubated at 37 C., 5% CO.sub.2 for 48 hours. After 48 hours post infection, the cells were tested for GFP expression by flow cytometry. Samples were analysed on a BD FACSCalibur using an Argon 488 Laser, gated for positivity against unstained HEK 293 cells. The level of GFP positive cells was then used to calculate virus titre.

Example 7: Lentivirus Production at Standard Titre

[0205] The most productive VSV G expressing cell lines and wild-type HEK 293 cells were seeded in DMEM media (10% FCS, 1% penicillin/streptomycin) into 10 cm dishes 24 hour prior to transfection so as to be at 80% confluent at the point of transfection.

[0206] At the time of transfection, DNA/PEI complexes were made up as follows:

[0207] For the VSV G line cell transfections, the following amounts of DNA/well were added to 2.5 ml of optimum per 10 cm dish in a master solution: [0208] 5 g Lentivirus GFP vector [0209] 5 g Rev plasmid [0210] 12 g Gag/pol plasmid.

[0211] An amount of branched PEI equivalent to 3 the total weight of DNA was added to another 2.5p1 of optimum per well in a separate tube.

[0212] Both solutions were filtered through a 0.2 m sterile filter, and then the PEI solution was added dropwise to the DNA solution.

[0213] The same protocol was used to make up a transfection complex for the transfection of the unmodified HEK 293 cells which also included 1.5 g of VSV G plasmid.

[0214] The transfection complexes were incubated for 20 minutes at room temperature in which time the cells were washed with optimum media, then 5 ml of each transfection complex was added to the respective 10 cm dishes. The cells were left to incubate at 37 C., 5% CO.sub.2 for 5 hours, after which time the media was changed for DMEM (10% FCS, 1% penicillin/streptomycin).

[0215] This virus containing supernatant was harvested at 48 hours and replaced with fresh media. The supernatant was again harvested at 72 hours post-transfection and stored at 4 C.

[0216] In order to calculate virus titre, wild type HEK 293 cells were seeded in DMEM (10% FCS, 1% penicillin/streptomycin) to a density of 90% in a 48 well plate 24 hours prior to infection. Four concentrations of harvested supernatant from each time point were used to infect cells in triplicate wells. Three five-fold serial dilutions were made into DMEM (10% FCS, 1% penicillin/streptomycin). Cells were infected by removing the overnight media, and adding 500ul of diluted supernatant.

[0217] Cells were incubated at 37 C., 5% CO.sub.2 for 48 hours. After 48 hours, samples were analysed on a BD FACSCalibur using an Argon 488 Laser, gated for positivity against unstained HEK 293 cells. The level of GFP positive cells was then used to calculate virus titre.

Example 8: Measurement of Cell Viability

[0218] To determine the effectiveness of incorporating apoptosis inhibitors into DNA constructs, plasmids were generated encoding the VSV G glycoprotein and the puromycin resistance gene with and without a range of apoptosis inhibitor genes (e.g. FIGS. 7-8). When DNA plasmids without apoptosis inhibitors were transfected into cells, colonies were formed at 3 g/ml and 5 g/ml puromycin. However, compared to cells transfected with DNA constructs containing apoptosis inhibiting genes, approximately 20-fold less colonies were formed. Those colonies that did grow, particularly at 5 g/ml puromycin, were both small, slow growing, and all colonies showed diffuse, ill-defined, membrane boundaries when growing in close proximity. Over a period of 6-8 weeks, these cells continued to grow slowly and were morphologically distinct from standard HEK 293 cells and those cell lines generated by simultaneously incorporating genes encoding apoptosis inhibitors. When sufficient cell numbers were available for lentivirus production, cell lines without apoptosis inhibitors were found to be significantly more sensitive to transfection mediated toxicity, perhaps due to the combined toxicity of both VSV G and the transfection process. These cells also produced virus at significantly lower levels compared to all of the cell lines generated by incorporating apoptosis inhibiting genes. These data demonstrate, in keeping with early observations (Pear et al., Proc. Natl. Acad. Sci. USA, Vol. 90, pp. 8392-8396, 1993) that cell lines can be generated without the addition of apoptosis inhibitors, but these cells grow poorly and have major limitations for retrovirus production.

[0219] Plasmids such as those shown in FIG. 8, where the VSV G coding region is separated from the resistance gene (e.g. for puromycin selection) by an IRES, ensuring that both are expressed in the same mRNA and thereby avoiding silencing of the toxic protein (eg. VSV G) by methylation, invariably allowed production of stable cell lines expressing higher levels of the toxic protein (e.g. VSV G).

Example 9: Use of CMV Promoter

[0220] The effect of a range of individual genes encoding apoptosis inhibitors, expressed using a strong CMV promoter, on the levels of luciferase expression observed in HEK293 cells is shown in FIG. 9. Luciferase was also regulated by a CMV promoter. The most effective genes included KSbcIL2, BHRF1, Vaccinia SP2 and ASFV5HL. Interestingly, XIAP was less effective when expressed from a CMV promoter.

Example 10: Use of SV40 Promoter

[0221] The effect of a range of individual genes encoding apoptosis inhibitors, expressed using an SV40 promoter (which is substantially weaker than a CMV promoter) on the levels of luciferase expression observed in HEK293 cells is shown in FIG. 10. Luciferase was regulated by a CMV promoter. The most effective genes included KSbcl2, XIAP, M11L and SV40 Large T antigen. BHRF1 was less effective when expressed at these relatively low levels.

Example 11: Use of Pairs of Apoptosis Inhibitor Genes

[0222] The effect of combining pairs of apoptosis inhibitor genes under the control of CMV and SV40 promoters, to give comparatively high and low levels of expression, respectively, in HEK293 cells is shown in FIG. 11. Luciferase expression was driven by the CMV promoter and was measured after 24 and 48 hours. The most effective combinations over 24 hours included highKSbc12+lowXIAP; highKSbc12+lowM11L; high BHRF1+lowKSbcl2. The most effective combinations over 48 hours included high BHRF1+lowXIAP; highASFV5HL+lowXIAP; and highKSbcl2+lowlAP1.

Example 12: Expression of Human IgG Antibody

[0223] Human IgG antibody expression in suspension HEK-293 cells using a specific apoptosis inhibitor combination, namely KSbcl2 and BHRF1 both under regulatory control of the CMV promoter, is shown in FIG. 12. Lanes 1: ladder. Lanes 2&3: competitor's plasmid at 12 g. Lanes 3&4: competitor's plasmid at 24 g. Lanes 15&16: plasmid containing a specific apoptosis inhibitor combination at 12 g. Lanes 17&18: plasmid containing a specific apoptosis inhibitor combination at 24 g. Apoptosis inhibitors allow the usage of higher DNA quantities without seeing the reduction in protein expression seen in lanes 4&5.

Example 13: Expression of Yellow Fever NS1 Secreted Protein

[0224] Yellow fever NS1 secreted protein expression was measured via ELISA after 3 days from transfected suspension HEK-293 cells. Sample A contains a standard CMV expression plasmid. Sample K contains a plasmid encoding KSbcl2 apoptosis inhibitor under regulatory control of the CMV promoter, alongside the CMV promoter-driven NS1 plasmid. The KSbcl2 gene significantly increased NS1 protein expression as shown in FIG. 13.

SEQUENCES

[0225]

TABLE-US-00001 (VSVG,VesicularStomatitisVirus) SEQIDNO:1 MKCLLYLAFLFIGVNCKFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHND LIGTALQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFT PSVEQCKESIEQTIKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLV DEYTGEWVDSQFINGKCSNYCPTVHNSTTWHSDYKVKGLCDSNLISMDITF FSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEM ADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSK IRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILS RMVGMISGTTTERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGML DSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFS SWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLG K (GAG-POL,HumanImmunodeficiencyVirus) SEQIDNO:2 MGARASVLSGGELDRWEKIRLRPGGKKKYKLKHIVVVASRELERFAVNPGL LETSEGCRQILGQLQPSLQTGSEELRSLYNTVATLYCVHQRIEIKDTKEAL DKIEEEQNKSKKKAQQAAADTGHSSQVSQNYPIVQNIQGQMVHQAISPRTL NAWVKVVEEKAFSPEVIPMFSALSEGATPQDLNTMLNTVGGHQAAMQMLKE TINEEAAEWDRVHPVHAGPIAPGQMREPRGSDIAGTTSTLQEQIGWMTNNP PIPVGEIYKRWIILGLNKIVRMYSPTSILDIRQGPKEPFRDYVDRFYKTLR AEQASQEVKNWMTETLLVQNANPDCKTILKALGPAATLEEMMTACQGVGGP GHKARVLAEAMSQVTNSATIMMQRGNFRNQRKIVKCFNCGKEGHIARNCRA PRKKGCWKCGKEGHQMKDCTERQANFLREDLAFLQGKAREFSSEQTRANSP TISSEQTRANSPTRRELQVVVGRDNNSLSEAGADRQGTVSFNFPQITLWQR PLVTIKIGGQLKEALLDTGADDTVLEEMSLPGRWKPKMIGGIGGFIKVRQY DQILIEICGHKAIGTVLVGPTPVNIIGRNLLTQIGCTLNFPISPIETVPVK LKPGMDGPKVKQWPLTEEKIKALVEICTEMEKEGKISKIGPENPYNTPVFA IKKKDSTKWRKLVDFRELNKRTQDFWEVQLGIPHPAGLKKKKSVTVLDVGD AYFSVPLDEDFRKYTAFTIPSINNETPGIRYQYNVLPQGWKGSPAIFQSSM TKILEPFRKQNPDIVIYQYMDDLYVGSDLEIGQHRTKIEELRQHLLRWGLT TPDKKHQKEPPFLWMGYELHPDKWTVQPIVLPEKDSWTVNDIQKLVGKLNW ASQIYPGIKVRQLCKLLRGTKALTEVIPLTEEAELELAENREILKEPVHGV YYDPSKDLIAEIQKQGQGQWTYQIYQEPFKNLKTGKYARTRGAHTNDVKQL TEAVQKITTESIVIWGKTPKFKLPIQKETWETVWVTEYVVQATWIPEWEFV NTPPLVKLWYQLEKEPIVGAETFYVDGAASRETKLGKAGYVTNRGRQKVVT LTDTTNQKTELQAIHLALQDSGLEVNIVTDSQYALGIIQAQPDKSESELVN QIIEQLIKKEKVYLAWVPAHKGIGGNEQVDKLVSAGIRKVLFLDGIDKAQD EHEKYHSNWRAMASDFNLPPVVAKEIVASCDKCQLKGEAMHGQVDCSPGIW QLDCTHLEGKVILVAVHVASGYIEAEVIPAETGQETAYFLLKLAGRWPVKT IHTDNGSNFTSTTVKAACVWVAGIKQEFGIPYNPQSQGVVESMNKELKKII GQVRDQAEHLKTAVQMAVFIHNFKRKGGIGGYSAGERIVDIIATDIQTKEL QKQITKIQNFRVYYRDSRDPLWKGPAKLLWKGEGAVVIQDNSDIKVVPRRK AKIIRDYGKQMAGDDCVASRQDED