Jefferiss Research Trust Laboratories, Wright Fleming Institute, Division of Medicine, Imperial College London, London W2 1PG, UK
Correspondence
Myra O. McClure
m.mcclure{at}imperial.ac.uk
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ABSTRACT |
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A figure showing the derivation of the constructs expressing the gag and pol genes is available as supplementary material in JGV Online.
Present address: Institute for Biomedical Research, Georg-Speyer-Haus, Paul-Ehrlich-Str. 4244, D-60596 Frankfurt/Main, Germany.
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MAIN TEXT |
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Hirata et al. (1996) showed that haematopoietic progenitor cells (HPCs), pre-stimulated into cycling by cytokines, were transduced similarly by PFV- and MLV-based vectors. However, more recent studies have shown that freshly isolated and hence quiescent (G0/G1) murine and human HPCs are transduced readily in vitro by both PFV (Vassilopoulos et al., 2001
; Josephson et al., 2002
) and SFV-1 (Zucali et al., 2002
) vectors in the presence of cytokines. Also, in vivo studies in which HPCs were transduced ex vivo and reintroduced into NOD/SCID mouse models showed efficient transgene expression in engrafted cells (Vassilopoulos et al., 2001
; Josephson et al., 2002
, 2004
; Zucali et al., 2002
; Leurs et al., 2003
).
The aim of this study was to compare the effect of growth arrest on the transduction of cell lines by MLV, PFV and HIV vectors at low titres and to assay the efficiency of these vectors on HPCs.
The MLV vectors were produced from the packaging cell lines FLY A4 LacZ 3 (Cosset et al., 1995) and FLY MFG NC (kindly provided by Professor Mary Collins). The resulting Moloney MLV (MoMLV) cores contain the LacZ and eGFP transgenes, respectively, driven by the MoMLV long terminal repeat (LTR) promoter, with the virions surrounded by the amphotrophic MLV envelope.
The HIV vector was made by transient transfection using the following constructs: either pH7G, which encodes eGFP under the control of the CMV promoter (Ikeda et al., 2003), or pH7nZ, which is the same as pH7G, but contains the LacZ transgene in place of eGFP, the HIV Gag-Pol construct, pSYNGP (Kotsopoulou et al., 2000
) and the VSV-G envelope construct, pRV67 (Mitrophanous et al., 1999
).
The pMD11 and pMD9 plasmids encode the PFV vector genome (Heinkelein et al., 2002). The pMD11 plasmid encodes LacZ and pMD9 encodes eGFP, both driven by the spleen focus-forming virus (SFFV) LTR promoter. pMD9 was adapted by exchanging the SFFV LTR for either a MoMLV LTR promoter [pMD9 (MoMLV)] or a CMV promoter [pMD9 (CMV)]. These vector constructs were co-transfected with plasmid pczHFVenv, which encodes the PFV envelope protein (Pietschmann et al., 1999
), and the packaging construct pKOgp1, which is derived from pCgp1 (Fischer et al., 1998
) and encodes the PFV Gag and Pol proteins under the control of the CMV promoter (see Supplementary Figure in JGV Online). Much of the U5 region and the leader sequence are absent, including the primer binding site and the RNA dimerization sequence (Erlwein et al., 1998
; Heinkelein et al., 1998
). The splice donor at position 51 (Muranyi & Flügel, 1991
) is retained to facilitate Pol expression from its spliced mRNA. To do this, the gag sequences at positions 12742915 (numbers are according to the PFV infectious clone, HSRV-2; Rethwilm et al., 1990
) were PCR-amplified by using high-fidelity Pwo polymerase (Roche). The forward primer sequence was 5'-TTTCCCTTGCTAAGGCCGCCGCCACCATGGCTTCAGGAAGTAATGTTGAAGAATATAAC-3' and included an optimized Kozak sequence (Kozak, 1986
) upstream of the start codon of the gag ORF to optimize translation. The sequence of the reverse primer was 5'-TTTGAGGTTGGTAAGTACGGGGTCA-3'. The amplicon was cloned between the EcoNI site in R and the SwaI site in the gag ORF of pCgp1, thereby deleting nt 10871273 in the HSRV-2 leader sequence.
The PFV and HIV vectors were produced by transient transfection with polyethylenimine (PEI) (Sigma). Briefly, 293T cells were seeded at 4x106 cells per plate in 10 ml Dulbecco's modified Eagle's medium (DMEM) supplemented with 10 % fetal calf serum (FCS), 1000 U penicillin ml1, 100 µg streptomycin ml1 and 2 mM L-glutamine (complete medium, CM) and incubated overnight at 37 °C. For the PFV vector, 5 µg pMD11, pMD9(MoMLV) or pMD9(CMV), 9 µg pKOGP and 3 µg pczHFVenv were co-transfected into cells and for the HIV vector, 8 µg pH7nZ or pH7G, 8 µg pSYNGP and 4 µg pRV67 were co-transfected. For each plate, plasmid DNA was diluted in 2·5 ml serum-free DMEM and added drop-wise with vortexing to 2·5 ml serum-free medium that contained 0·4 µM PEI. A PEI/DNA precipitate was formed after incubation at room temperature for 20 min and was added in full to fresh medium, which replaced the medium on the 293T cells prior to incubation for 45 h at 37 °C. The medium was changed to CM and the cells were incubated for a further 48 h at 32 °C. Vector supernatant was filtered through 0·45 µm filters (Millipore) and concentrated by ultracentrifugation at 50 000 g for 90 min at 4 °C. To produce MLV vector, FLY A4 LacZ 3 and FLY MFG NC cells were cultured to 70 % confluency. The medium was changed, the cells were incubated at 32 °C for 2 days and the supernatant was harvested and filtered. All vector stocks were titrated on D17 cells. Briefly, D17 cells were seeded at 3x104 cells per well and incubated at 37 °C overnight, then 250 µl vector (diluted serially tenfold to 105) was added. Transduced cells were incubated for 3 days at 37 °C and fixed in 4 % formaldehyde; -galactosidase expression was detected by the insoluble X-Gal assay (Bieniasz et al., 1995
) and eGFP expression was detected by fluorescence microscopy.
The effect of cell-cycle arrest on transduction by three LacZ-expressing retrovirus vectors was assessed on HT1080, D17, HeLa and TE671 cells. Transduction was assayed as described for vector titration. Treatment with 5 µM aphidicolin, a reversible inhibitor of eukaryotic DNA synthesis, for 24 h prior to transduction caused cell-cycle arrest at G1/S in all cell lines, confirmed by flow cytometry using propidium iodide (Bieniasz et al., 1995). Cell viability remained >90 %, as assessed by Trypan blue exclusion. For each cell line and vector, the number of G1/S-arrested cells expressing LacZ was given as a percentage of the number that were observed in cycling cells.
Whilst the MLV vector failed to transduce any cells that were not dividing, the HIV and PFV vectors transduced different cells at quite different efficiencies: D17 (30 and 0·6 %, respectively), HeLa (21 and 1·4 %), HT1080 (70 and 0·3 %) and TE671 (32 and 10 %).
Transduction efficiency in HT1080 cycling cells was given a reference value of 100 %, to which the efficiency in arrested cells could be compared [Fig. 1a (A)]. Cells arrested in G1/S phase, exposed to LacZ-expressing vector for 4 h and maintained under similar conditions for another 48 h, poorly expressed the transgene following transduction by MLV and PFV vectors [Fig. 1a (B)
]. When, on changing the medium, the cells were allowed to divide during the 4 h transduction and thereafter, transduction levels by PFV vector were comparable to those of HIV vector and threefold higher than those of MLV [Fig. 1a (C)
]. When transduced cells were arrested during transduction before release (4 h), PFV vector transduction was slightly lower than that of HIV [Fig. 1a (D)
]. When transduced cells were arrested for a further 24 h before release, the PFV vector again facilitated higher levels of transgene expression than the MLV vector, although this was reduced compared to the HIV vector [Fig. 1a (E)
]. When cells in G1/S arrest were exposed to mitomycin C (at 10 µg ml1 for 30 min pre-transduction) to prevent cycling through M phase following release from aphidicolin, vector titres were reduced to <0·05 % (MLV), 2 % (PFV) and 70 % (HIV) of the titres that were obtained in untreated cells [Fig. 1a (F)
]. Cells arrested irreversibly in G2 by exposure to 9000 rad (=90 Gy)
-irradiation from a 137Cs source prior to seeding were transduced by PFV (1 %) and better by HIV (48 %), but not by MLV (<0·05 %) [Fig. 1a (G)
].
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Haematopoietic progenitor CD34+ cells were isolated from cord blood by magnetic microbead selection (Miltenyi Biotech). RetroNectin-coated 24-well plates were pre-incubated with 500 µl retrovirus vector supernatant at 3x106 ml1 (titrated on D17 cells) for 2 h at 37 °C. CD34+ cells were added, to give a final concentration of 2x105 cells ml1, to X-Vivo 10 medium (Biowhittaker) supplemented with the cytokines interleukin 3 (IL3; 20 ng ml1), IL6 (20 ng ml1), stem cell factor (SCF; 100 ng ml1) and Flt-3 ligand (Flt-3; 100 ng ml1) (Tebu). Cells and vector supernatant were centrifuged in the 24-well plate and incubated for 16 h at 37 °C, after which a further 500 µl vector, supplemented with cytokines, was added to the cells. Cells were harvested and assayed for -galactosidase activity 48 h after the second transduction. Mean vector transduction rates were: HIV, 48 %; PFV, 37 %; and MLV, 56 % (Fig. 2
), reflecting differential dependence on host-cell division. Freshly isolated CD34+ cells are typically in the G0/G1 phase of the cell cycle and can be stimulated into division by cytokines. Our results are consistent with the premise that HIV vector delivery is largely independent of cellular mitosis, whereas MLV fails to transduce cells without prior cytokine stimulation (Uchida et al., 1998
) and PFV can infect quiescent cells, but requires cell division for integration and transgene expression (Trobridge & Russell, 2004
).
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Other factors besides NLS sequences could account for transduction differences in retrovirus vectors. Reverse transcription of PFV occurs mainly during PFV particle formation and the virions contain virus RNA and DNA (Yu et al., 1999); hence, the virions could be more stable. Our results demonstrate that the longer the time between vector entry and cell division, the lower the likelihood of successful integration. This is more marked for MLV than for PFV, suggesting that PFV is more stable intracellularly; it is possible that the PICs of foamy viruses are less susceptible to degradation by cellular nucleases than those of MLV.
It is clear that foamy viruses allow infection of non-dividing cells at a level intermediate between onco- and lentivirus vectors, but the integration efficiency remains significantly lower than that of lentiviruses unless cells are allowed to pass through mitosis. The data presented in this paper confirm our own findings on wild-type foamy viruses, as well as those published recently by Trobridge & Russell (2004), but are contrary to the findings of Mergia et al. (2001)
, possibly for reasons alluded to above. Hence, our data are likely to end the controversy that surrounds the question of whether PFV and vectors derived from the virus are likely to be effective in gene therapy targeted to end-differentiated cells.
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ACKNOWLEDGEMENTS |
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Received 21 April 2004;
accepted 1 July 2004.