REVERSE GENETICS USING NON-ENDOGENOUS POL I PROMOTERS

Abstract

Expression of a transgene is driven in a host cell using a pol I promoter which is not endogenous to an organism from the same taxonomic order from which the host cell is derived.

Claims

1. A host cell comprising at least one expression construct encoding a viral RNA molecule, wherein expression of the viral RNA molecule from the construct is controlled by a pol I promoter which is not endogenous to the host cell's taxonomic order.

2. A cell having at least one endogenous pol I promoter which control(s) expression of endogenous rRNA and at least one non-endogenous pol I promoter which control(s) expression of a viral RNA or the complement thereof.

3. A method for producing a recombinant virus, comprising a step of growing the cell of claim 1 under conditions where the viral RNA molecule is expressed in order to produce virus.

4. A method of preparing a virus, comprising steps of: (i) producing a recombinant virus by the method of claim 3; (ii) infecting a culture host with the virus obtained in step (i); (iii) culturing the host from step (ii) to produce further virus; and (iv) purifying virus obtained in step (iii).

5. A method for preparing a vaccine, comprising steps of (a) preparing virus by the method of claim 4 and (b) preparing vaccine from the virus.

6. The cell of claim 1, wherein the pol I promoter is a primate pol I promoter and the cell is a non-primate cell.

7. The cell of claim 1, wherein the pol I promoter is a non-canine pol I promoter and the cell is a canine cell.

8. The cell of claim 7 wherein the pol I promoter is a human pol I promoter and the cell is a canine cell.

9. The cell of claim 8 wherein the cell is an MDCK cell.

10. The cell of claim 9 wherein the MDCK cell is cell line MDCK 33016 (DSM ACC2219).

11. The cell of claim 1, wherein the cell includes at least one bidirectional expression construct

12. The cell of claim 1, wherein the expression construct is an expression vector or a linear expression construct.

13. The cell of claim 1, wherein the virus is a segmented virus.

14. The cell of claim 1, wherein the virus is a non-segmented virus.

15. The cell of claim 1, wherein the virus is a negative-strand RNA virus.

16. The cell of claim 15, wherein the virus is influenza virus.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0149] FIG. 1 illustrates the expression construct which was used for assaying pol I promoter activity with a luciferase reporter.

[0150] FIG. 2 shows the full-length (FL) human pol I promoter sequence (SEQ ID NO: 1). The pHW2000 human Pol I Promoter sequence (SEQ ID NO: 2; short human pol I promoter)) within the full-length sequence is shown in underlined fonts. The arrow indicates the transcriptional start site.

[0151] FIG. 3 shows the full-length (FL) canine pol I promoter sequence (NW_878945; SEQ ID NO: 3). The SHORT promoter sequence within the full-length promoter sequence is shown in underlined capital fonts (SEQ ID NO: 5); the MID promoter sequence within the full-length promoter sequence is shown in underlined capital fonts and bold lowercase fonts (SEQ ID NO: 4);

[0152] FIG. 4 shows canine pol I promoter activity in MDCK cells. The solid grey columns show the results with the FL canine pol I promoter, the cross-hatched columns show the results with the MID canine promoter and the dotted columns show the results with the SHORT canine pol I promoter. A indicates LUC and viral polymerase, B indicates LUC and infection (MOI=0.05), C indicates LUC and D is no DNA. The y-axis indicates relative light units (RLU).

[0153] FIG. 5 shows human pol I promoter activity in MDCK 33016 cells. The solid grey columns show the results with the human pol I promoter, the cross-hatched columns show the results with the FL canine pol I promoter, the dotted columns show the results with the MID canine pol I promoter and the vertically hatched columns show the results with the SHORT canine pol I promoter. A indicates LUC and viral polymerase, B indicates LUC and infection (MOI=0.05) and C indicates LUC. The y-axis indicates relative light units (RLU).

[0154] FIG. 6 shows a comparison of the activity of the FL and SHORT human pol I promoter and the full-length canine pol I promoter in MDCK cells 33016 cells. The solid grey columns show the results with the full-length human promoter, the hatched columns show the results with the full-length canine promoter and the dotted columns show the results with the short human pol I promoter. A indicates LUC+polymerase, B indicates LUC+infection and C shows LUC only. The y-axis indicates relative light units (RLU).

[0155] FIG. 7A shows the activity of the human pol I promoter (dotted columns) and the canine pol I promoter (cross-hatched columns) in MDCK 33016 cells. X indicates LUC+polymerase, Y indicates LUC+infection and Z shows LUC only. The y-axis indicates relative light units (RLU).

[0156] FIG. 7B shows the activity of the human pol I promoter (dotted columns) and the canine pol I promoter (cross-hatched columns) in MDCK ATCC cells. X indicates LUC+polymerase, Y indicates LUC+infection and Z shows LUC only. The y-axis indicates relative light units (RLU).

[0157] FIG. 8 shows a western blot analysis of M and NP proteins in cell lysates after virus rescue.

[0158] FIG. 9 shows results of a focus-forming assay using supernatant from cells infected with reverse genetics constructs.

[0159] FIG. 10 shows an alignment of DNA sequences of human and canine pol I promoters (SEQ ID NOs 1 and 3, respectively).

[0160] FIG. 11A shows the expression levels of a reporter transgene under control of the human pol I (hPolI) promoter or canine pol I promoter (cPolI) in MDCK ATCC, MDCK 33016-PF and 293T cells. The black columns represent the results with 293T cells, the white columns show the results with MDCK 33016-PF and the cross-hatched columns represent the results with MDCK ATCC cells.

[0161] FIG. 11B compares the transfection efficiency in human and canine cells. The y-axis in both graphs indicates relative light units (RLUs).

[0162] FIG. 12A shows the rescue of the A/Puerto Rico/8/34 influenza strain by human poll promoter-based reverse genetics in MDCK ATCC, MDCK 33016-PF and 293T cells in the presence (white columns) and absence (black columns) of the TMPRSS2 helper plasmid and with addition of feeder cells. The y-axis represents the virus titre (ffu/mL).

[0163] FIG. 12B shows the rescue of the A/Puerto Rico/8/34 influenza strain by human poll promoter-based reverse genetics in MDCK ATCC, MDCK 33016-PF and 293T cells in the presence (white columns) and absence (black columns) of the TMPRSS2 helper plasmid and without addition of feeder cells. The y-axis represents the virus titre (ffu/mL).

[0164] FIG. 13 compares the rescue of the A/Puerto Rico/8/34 influenza strain by human or canine pol I-driven reverse genetics in MDCK 33016-PF cells. The black columns show the results in the absence of the TMPRSS2 helper plasmid and the white bars show the result in the presence of the TMPRSS2 helper plasmid. The y-axis represents the virus titre (ffu/mL).

BRIEF DESCRIPTION OF SEQUENCE LISTING

[0165] SEQ ID NO: 1 is the full-length (FL) human pol I promoter sequence

[0166] SEQ ID NO: 2 is the pHW2000 human Pol I promoter sequence

[0167] SEQ ID NO: 3 is the full-length (FL) canine pol I promoter sequence

[0168] SEQ ID NO: 4 is the MID canine pol I promoter sequence

[0169] SEQ ID NO: 5 is the SHORT canine pol I promoter sequence

[0170] SEQ ID NO: 6 is the HA sequence from A/California/04/09

[0171] SEQ ID NO: 7 is the HA sequence from A/Chile/1/1983

[0172] SEQ ID NO: 8 is the NA sequence from A/California/04/09

[0173] SEQ ID NO: 9 is the NA sequence from A/Chile/1/1983

MODES FOR CARRYING OUT THE INVENTION

The Human Pol I Promoter is Active in Human as Well as Canine Cells.

[0174] In order to assess the activity of the pol I promoter in non-endogenous host cells, MDCK cells were transfected with an expression construct which allows expression of a luciferase (luc) RNA in antisense direction under control of a 487 bp fragment of the human pol I promoter or various fragments of the canine pol I promoter (as shown in FIG. 3). The expressed RNA can be transcribed into mRNA by a viral polymerase and subsequently be translated into luc protein. Thus, cells expressing the transgene can be easily identified by assaying for luciferase activity. In order for the assay to work it is necessary to provide viral polymerase. This can be achieved by co-transfecting the cell with expression constructs which encode the viral polymerase or, alternatively, infecting the transfected cell with a helper virus. The assay is illustrated in FIG. 1.

[0175] FIG. 11A shows that the human pol I promoter is able to drive expression of the transgene in MDCK ATCC cells and also in MDCK 33016-PF cells with the same efficiency as the canine pol I promoter. The expression levels of the transgene with the human pol I promoter in MDCK ATCC cells are even higher than those observed in human 293T cells. In order to confirm that the transfection efficiency of the tested cell types are comparable, they were transfected with a construct containing a luciferase gene under control of a CMV promoter. The level of luciferase activity was measured. The results are shown in FIG. 11B and confirm that the transfection efficiency of the tested cells is comparable.

[0176] FIG. 4 shows that all tested fragments of the canine pol I promoter can drive expression of the luc transgene in MDCK cells. Furthermore, FIG. 5 demonstrates that the full-length human pol I promoter is able to drive expression of the transgene in MDCK cells and is even more efficient than the canine pol I promoter.

[0177] In order to further define the region of the human pol I promoter which is necessary to drive expression of the transgene, the experiment was repeated with a fragment of the human pol I promoter as shown in FIG. 2 (short pol I). It was found that, while the full length pol I promoter is more active, the full-length as well as the short human pol I promoter are active in MDCK cells (FIG. 6).

[0178] The constructs containing the human and canine pol I promoter sequences were further transfected into MDCK from ATCC and MDCK 33016 cells [18] in order to determine whether the activity of the human pol I promoter is restricted to a certain cell line. As shown in FIG. 7A and FIG. 7B, the human pol I promoter was able to drive expression of the transgene in both cell types but the expression was more efficient in MDCK 33016 cells. Rescuing influenza virus from MDCK cells using human pol I promoter

[0179] The efficiency of influenza virus rescue using the human pol I promoter was compared in MDCK and 293T cells. The influenza viral genome was cloned into pHW2000 expression vectors [69]. This vector contains a fragment of the human pol I promoter which was shown to be active in MDCK cells (see FIG. 5). In particular, the following vectors were used: pHW-WSN PA (0.534 g/1); pHW-WSN PB1 (0.432 g/1); pHW-WSN PB2 (0.357 g/1); pHW-WSN NP (0.284 g/1); pHW-WSN NS (0.217 g/1); pHW-WSN M (0.232 g/1); pHW-WSN HA (0.169 g/1); pHW-WSN NA (0.280 g/1) and pcDNA-TMPRSS (0.775 g/1; encoding serine protease). Protein-coding genes were controlled by a cytomegalovirus (CMV) promoter.

[0180] For the virus rescue, 293T cells were seeded at a density of 510.sup.6 cells/well in 6-well dishes with 2 ml of Dulbecco's Modified Eagle Medium (DMEM) with 10% FCS. MDCK cells were plated at 0.310.sup.6 cell/well in 6-well dished with 2 ml of medium. The cells were incubated overnight at 37 C. and were transfected when they had reached a confluency of 50-80%.

[0181] 293T and MDCK cells were transfected using FuGENE 6 Transfection Reagent (Roche Cat.#11988387001) and Lipofectamine LTX Plus Reagent (Invitrogen Cat.#15338-100), respectively. The cells were transfected with 1 g of each vector in accordance with the following protocols. For FuGENE 6, the reagent (3 l of FuGENE/g DNA) was diluted in 730 serum-free medium (without antibiotics), mixed gently and incubated at room temperature for 5 minutes. Afterwards the DNA was added to each to the diluted FuGENE, mixed gently and incubated at room temperature for at least 15 minutes. The DNA/FuGENE complex was added drop wise to the 293T cells without removing the growth medium and the cells were incubated at 37 C. for 24 hours.

[0182] For transfection with lipofectamine, the reagent (250) was diluted in 500 l serum-free medium and incubated at room temperature for 5 minutes. The DNA was added and the mixture was incubated at room temperature for 30 minutes. Following incubation, 500 l of serum-free medium was added drop wise to the transfection reagent after the growth medium had been removed from the cells. The cells were subsequently incubated at 37 C. for 24 hours. 24 hours after transfection, the medium was changed.

[0183] Two days after infection, the supernatant from the cells was collected by centrifugation at 1000 rpm for 5 minutes. The virus collected in the supernatant was used for Focus-Forming Assays. Furthermore, the infected cells were lysed and used for Western Blot analysis.

Western Blot Analysis

[0184] The 293T and MDCK cells were lysed and subjected to Western Blot analysis in accordance with standard protocols. Antibodies against the M and NP protein were used to detect these proteins on the membrane. Antibodies against S6 were used as a loading control. The lanes labelled as WSN were loaded with proteins from the rescued virus. The lanes labelled M and NP contain recombinant M and NP proteins as a control. As these recombinant proteins were expressed from a different gene they migrate slightly slower in the gel.

[0185] The results of the analysis are shown in FIG. 8 where it is evident that the expression construct under control of the human pol I promoter allows viral rescue in 293T as well as in MDCK cells.

Focus-Forming Assays

[0186] Uninfected MDCK cells were plated at a density of 6.2510.sup.4 cells/well in 48 well plates in 500 l of DMEM with 10% FCS. The next day cells were infected with viruses in a volume of 100-1500 for 2 hours at 37 C. The cells were thereby infected with various dilutions of the virus. Two hours post-infection, the medium was aspirated and 500 l of DMEM with 10% FCS was added to each well. The cells were incubated at 37 C. until the next day.

[0187] 24 hours after infection, the medium was aspirated and the cells washed once with PBS. 500 l of ice-cold 80% acetone in PBS was added to each well followed by incubation at 4 C. for 30 minutes. The acetone mix was aspirated and the cells washed once with PBST (PBS+0.1% Tween). 500 l of 2% BSA in PBS was added to each well followed by incubation at room temperature (RT) for 30 minutes. 500 l of a 1:6000 dilution of anti-NP was added in blocking buffer followed by incubation at RT for 2 hours. The antibody solution was aspirated and the cells washed twice with PBST. Secondary antibody (goat anti mouse) was added at a dilution 1:2000 in 500 l blocking buffer and the plate was incubated at RT for 2 hours. The antibody solution was aspirated and the cells washed three times with PBST. 500 l of KPL True Blue was added to each well and incubated for 10 minutes. The reaction was stopped by aspirating the True-Blue and washing once with dH.sub.2O. The water was aspirated and the cells left to dry.

[0188] The results of the assay are shown in FIG. 9 which demonstrates clearly that infectious virus was obtained from 293T as well as MDCK cells.

[0189] Virus rescue of the A/Puerto Rico/8/34 influenza strain using a human pol I reverse genetics system was also tested in MDCK ATCC, MDCK 33016-PF and 293T cells as described in reference 70. Experiments were performed in which the virus was rescued in the presence and absence of the helper plasmid TMPRSS2 which encodes a serine protease. Furthermore, the viral rescue was performed with and without the addition of feeder cells 24 hours after the viral rescue. The results are shown in FIG. 12A and FIG. 12B and demonstrate that efficient viral rescue could be achieved in MDCK cells under various conditions using the human pol I promoter.

[0190] To compare whether the efficiency of viral rescue in MDCK 33016-PF using a human pol I promoter is comparable with the rescue using a canine pol I promoter, the cells were transfected with a human pol I RG system or a canine pol I RG system as described in reference 70. The experiments were performed in the presence and absence of the TMPRSS helper plasmid. The results (FIG. 13) demonstrate that the A/Puerto Rico/8/34 strain was rescued with comparable efficiency to the canine pol I system when the human pol I system was used.

[0191] It will be understood that the invention has been described by way of example only and modifications may be made whilst remaining within the scope and spirit of the invention.

REFERENCES

[0192] [1] WO2007/002008 [0193] [2] WO2007/124327 [0194] [3] Koudstaal et al. (2009) Vaccine 272588-2593 [0195] [4] WO2009/000891 [0196] [5] Sambrook et al, Molecular Cloning: A Laboratory Manual, 2 ed., 1989, Cold Spring Harbor [0197] Press, Cold Spring Harbor, N. Y [0198] [6] Racaniello and Baltimore (1981) Science 214:916-919 [0199] [7] Kaplan et al. (1985) Proc Natl Acad Sci USA 82: 8424-8428 [0200] [8] Fodor et al. (1999) J. Virol 73(11):9679-9682 [0201] [9] Hoffmann et al. (2002) Proc Natl Acad Sci USA 99: 11411-11416 [0202] [10] Kobayashi et al. (2007) Cell Host Microbe 19; 1(2):147-57 [0203] [11] Stech et al. (2008) Nucleic Acids Res 36(21):e139 [0204] [12] Kistner et al. (1998) Vaccine 16:960-8 [0205] [13] Kistner et al. (1999) Dev Biol Stand 98:101-110 [0206] [14] Bruhl et al. (2000) Vaccine 19:1149-58 [0207] [15] Pau et al. (2001) Vaccine 19:2716-21 [0208] [16] http://www.atcc.org/[17] [0209] http://locus.umdnj.edu/[18] [0210] WO97/37000 [0211] [19] Brands et al. (1999) Dev Biol Stand 98:93-100 [0212] [20] Halperin et al. (2002) Vaccine 20:1240-7 [0213] [21] EP-A-1260581 (WO01/64846) [0214] [22] WO2006/071563 [0215] [23] WO2005/113758 [0216] [24] WO97/37001 [0217] [25] Neumann et al. (2005) Proc Natl Acad Sci USA 102: 16825-9 [0218] [26] WO02/28422 [0219] [27] WO02/067983 [0220] [28] WO02/074336 [0221] [29] WO01/21151 [0222] [30] WO02/097072 [0223] [31] WO2005/113756 [0224] [32] Vaccines. (eds. Plotkin & Orenstein). 4th edition, 2004, ISBN: 0-7216-9688-0 [0225] [33] Huckriede et al. (2003) Methods Enzymol 373:74-91 [0226] [34] Treanor et al. (1996) J Infect Dis 173:1467-70 [0227] [35] Keitel et al. (1996) Clin Diagn Lab Immunol 3:507-10 [0228] [36] Herlocher et al. (2004) J Infect Dis 190(9):1627-30 [0229] [37] Le et al. (2005) Nature 437(7062):1108 [0230] [38] WO2008/068631 [0231] [39] Gennaro (2000) Remington: The Science and Practice of Pharmacy. 20th edition, ISBN: 0683306472 [0232] [40] Banzhoff (2000) Immunology Letters 71:91-96 [0233] [41] Nony et al. (2001) Vaccine 27:3645-51 [0234] [42] EP-B-0870508 [0235] [43] U.S. Pat. No. 5,948,410 [0236] [44] WO2007/052163 [0237] [45] WO2007/052061 [0238] [46] WO90/14837 [0239] [47] Podda & Del Giudice (2003) Expert Rev Vaccines 2:197-203 [0240] [48] Podda (2001) Vaccine 19: 2673-2680 [0241] [49] Vaccine Design: The Subunit and Adjuvant Approach (eds. Powell & Newman) Plenum Press 1995 (ISBN 0-306-44867-X) [0242] [50] Vaccine Adjuvants: Preparation Methods and Research Protocols (Volume 42 of Methods in Molecular Medicine series). ISBN: 1-59259-083-7. Ed. O'Hagan [0243] [51] WO2008/043774 [0244] [52] Allison & Byars (1992) Res Immunol 143:519-25 [0245] [53] Hariharan et al. (1995) Cancer Res 55:3486-9 [0246] [54] US-2007/014805 [0247] [55] US-2007/0191314 [0248] [56] Suli et al. (2004) Vaccine 22(25-26):3464-9 [0249] [57] WO95/11700 [0250] [58] U.S. Pat. No. 6,080,725 [0251] [59] WO2005/097181 [0252] [60] WO2006/113373 [0253] [61] Potter & Oxford (1979) Br Med Bull 35: 69-75 [0254] [62] Greenbaum et al. (2004) Vaccine 22:2566-77 [0255] [63] Zurbriggen et al. (2003) Expert Rev Vaccines 2:295-304 [0256] [64] Piascik (2003) J Am Pharm Assoc (Wash D.C.). 43:728-30 [0257] [65] Mann et al. (2004) Vaccine 22:2425-9 [0258] [66] Halperin et al. (1979) Am J Public Health 69:1247-50 [0259] [67] Herbert et al. (1979) J Infect Dis 140:234-8 [0260] [68] Chen et al. (2003) Vaccine 21:2830-6 [0261] [69] Hoffmann et al. (2000) Proc Natl Acad Sci USA 97:6108 [0262] [70] Suphaphiphat et al. (2010) J. Virol. 84(7) 3721-3725