Retrovirus vector production and transduction: modulation by the cell cycle

Alla Dolnikov1,2, Simon Wotherspoon3,4, Michelle Millington1,2 and Geoff Symonds1,2,4,5

1 Department of Clinical Pharmacology and Toxicology, St Vincent's Hospital, Darlinghurst, NSW 2010, Australia
2 School of Physiology and Pharmacology, The University of New South Wales, Sydney, NSW 2052, Australia and Children's Cancer Institute, Randwick, Sydney, NSW 2031, Australia
3 Department of Biotechnology, The University of New South Wales, Sydney, NSW 2053, Australia
4 Johnson and Johnson Research Laboratories, The Australian Technology Park, Eveleigh, NSW 1430, Australia
5 Department of Medicine, St Vincent's Hospital, Darlinghurst, NSW 2010, Australia

Correspondence
Geoff Symonds
(at Johnson and Johnson Research Locked Bag 4555 Strawberry Hills NSW 2012 Australia)
gsymonds{at}medau.jnj.com


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In this study, the cell cycle modulation of retrovirus vector production and transduction was analysed. Retrovirus vector expression was found to be similar in all phases of the cell cycle and, in contrast to some other virus promoters shown previously to be upregulated by G2/M arrest, Moloney murine leukaemia virus LTR-driven expression was upregulated neither by G2/M growth arrest nor by G1/S growth arrest. In contrast, cultures enriched for S phase cells produced more infectious virions, apparently by modulation of stages consequent to provirus expression. In terms of retrovirus transduction, limitations appear to be slow progression through the cell cycle and short half-life of the virus. Synchronization of cells prior to mitosis can increase transduction efficiency. Cell cycle modulation can be used to modify retrovirus vector production and transduction and can allow short transduction periods.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Retrovirus vectors are able to deliver a particular gene of interest to target cells and have been used commonly in experimental and clinical settings (Romano et al., 1999). Current methods for the generation of relatively high titre, helper-free retrovirus stocks utilize the creation of stable producer cell lines or, less frequently, transient producer cell lines that produce retrovirus vectors (Pear et al., 1993). Virus titres produced from commonly used retrovirus packaging cell lines tend to range from 105 to 107 infectious units ml-1, with variations related to the packaging cell line and vector used (Pear et al., 1993; Abonour et al., 2000; Malech, 2000; Pollok et al., 2001). Both harvest and transduction protocols are important, particularly for target cell types such as T-lymphocytes and human haematopoietic stem/progenitor cells (HSCs) (Dick, 2000).

Two main steps can be denoted in retrovirus vector production: (i) transcription of the provirus DNA and (ii) the formation of virus particles and their subsequent release from the producer cell (Miller, 1992). Moloney murine leukaemia virus (MoMLV)-based retrovirus vectors are used widely for exogenous gene expression in mammalian cells (Hawley et al., 1994). It has been shown using gene expression constructs that the human immunodeficiency virus type 1 (HIV-1) Vpr protein activates HIV-1 LTR-driven expression and that this activation correlates with Vpr-induced G2/M growth arrest (Goh et al., 1998). This same report showed that this resulted in more effective infection by vpr-containing HIV variants (Goh et al., 1998). Other reports showed that expression from virus promoters, such as the simian virus 40 (SV40) promoter and the HIV-1, human T-cell lymphotropic virus type 1 (HTLV-1) and Rous sarcoma virus LTRs, was induced by 2-aminopurine, an agent that also arrests cells at the G2/M phase (Maio et al., 1995). In the present study, we used chemicals to modulate the cell cycle and different times of harvest to examine cell cycle-dependent retrovirus vector expression, production and transduction.

Successful vector integration following retrovirus entry is dependent on mitosis and is limited by the rate of intracellular decay of internalized vectors (Miller et al., 1990; Roe et al., 1993; Lewis & Emerman, 1994). For MoMLV-derived retrovirus vectors, the intracellular half-life is in the range of 5·5 to 7·5 h (Andreadis et al., 1997). We hypothesized that retrovirus transduction of target cells that are in late S or G2/M phase will result in successful vector integration, while cells at the G1 phase at the time of virus exposure, normally comprising 40–60 % of an exponentially growing cell population, are less likely to be transduced. It is relevant that even for relatively rapidly cycling cells, such as NIH 3T3 cells, which have a doubling time of approximately 14 h, it takes more than 11 h for early G1 cells to reach mitosis (Karn et al., 1989). It has been speculated that greater than 24 h of exposure of target cells to virus (with addition of fresh virus every 6 h) is required to reach a maximum degree of infection (Morgan et al., 1995; Pollok et al., 2001). We sought to determine the effects of modulation of the cell cycle on retrovirus vector production and the efficiency of retrovirus transduction.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Vectors.
Two retrovirus vectors were employed. The GFP-containing vector, LGL, was produced by cloning GFPS65T (Clontech) downstream of the packaging site and the portion of gag remaining in the L9XL retrovirus vector. L9XL was constructed by the deletion of neo from LNL6 (Miller, 1992) and the insertion of a polylinker with multiple cloning sites. LNGFR was constructed by cloning a SalI–BamHI fragment encoding truncated human nerve growth factor receptor (NGFR) cDNA from the LNGFR vector pBlueScript KS+NGFR (generously provided by A. Bower, UNSW, Sydney, Australia) into L9XL. The expression plasmid pGFPtpzC (Canberra-Packard) was used to examine SV40 promoter-driven GFPtpz expression.

Virus-producing cell lines.
The amphotropic PA317 and GALV-pseudotyped PG13 packaging cell lines were transfected with the retrovirus DNA vectors to generate PA317/LGL, PG13/LGL and PA317/NGFR virus-producing cell lines, as described previously (Shounan et al., 1996). Transduced cells were identified by flow cytometry (GFP fluorescence or fluorescent antibody staining for NGFR) using a FACSort (Becton Dickinson). Clonal producer cell lines were derived by dilutional cloning. The strongest virus producers were chosen based on the percentage of GFP expression at 48 h after infection of NIH 3T3 or human 293 cells.

Cell culture.
Retrovirus vector-producing cells PA317 and PG13, as well as NIH 3T3, 293 and Jurkat cells, were maintained in DMEM (Gibco-BRL) supplemented with 10 % FBS, 2 mM L-glutamine, 100 U penicillin ml-1 and 0·1 mg streptomycin ml-1. Human leukaemic TF-1 cells were maintained in RPMI 1640 medium (Gibco-BRL) supplemented with 10 % FBS, 2 mM L-glutamine, antibiotics and 2 ng granulocyte macrophage colony-stimulating factor ml-1. All cell lines were obtained from ATCC.

To arrest cell growth, cells were treated for 16 h with 10 µg aphidicolin ml-1 (Sigma) to produce a G1/S block or 0·1 µg nocodazole ml-1 (Sigma) to produce a G2/M block. In addition, 10 mM 2-aminopurine (Sigma) was tested as a potential inhibitor of cell growth at the G2/M phase (Maio et al., 1995). Cells were released from these blocks, thus inducing synchronization, by removing the inhibitor-containing medium, washing three times in PBS and adding fresh medium without the inhibitor.

Preparation of retrovirus vectors and virus infection.
Retrovirus-producing cells were generally plated at 70 % confluency and vector-containing medium (VCM) was collected at the time periods indicated. Once harvested, VCM was filtered through a 0·45 µM filter and placed onto target cells at 5x105 cells ml-1 in the presence of 8 µg polybrene ml-1 for varying lengths of time. In some experiments, cells were infected using recombinant fibronectin fragment CH29 (RetroNectin, Takara Biomedicals). Cells were treated with RetroNectin according to the manufacturer's protocols. Cells were analysed by flow cytometry to determine reporter gene expression at 48 h and 5 days following infection.

Retrovirus vector half-life measurements.
To determine the half-life of infectious virions, supernatants were incubated at 37 or 32 °C for varying lengths of time. Their consequent titres were then measured on target cells at several time points. Retrovirus vectors produced by PG13/LGL and PA317/LGL cells were tested.

Flow cytometric analysis of reporter gene expression and cell cycle status.
In all experiments, live cells were gated using forward scatter versus side scatter plots. GFP expression was detected in the FL1 channel without any further manipulation of the cells. Cell surface NGFR was detected using indirect immunofluorescent labelling. Cells were incubated with monoclonal NGFR antibody or IgG control (both from Boehringer Mannheim) for 1 h at room temperature, washed three times by centrifugation (1000 r.p.m. for 5 min) in PBS and incubated for a further 30 min with a secondary anti-mouse FITC-conjugated IgG (Boehringer Mannheim). Fluorescence related to NGFR expression was measured in the FL1 channel.

For cell cycle analysis, cells were fixed overnight in cold 70 % ethanol and then incubated for 1 h at room temperature with 50 µg propidium iodide (PI) ml-1 in the presence of 500 U RNase ml-1. NGFR expression was analysed in the FL2 channel by gating G1, S and G2/M cells based on their DNA content. Cell cycle profiles were based on data from 10 000 cells and displayed as a frequency histogram of PI fluorescence (FL2). Analyses were conducted after gating out cellular debris and aggregates. Cells in the G1 phase were identified as those with a DNA content of 2 N, cells in the S phase as those with a DNA content ranging from 2 to 4 N and cells in the G2/M phase as those with a DNA content of 4 N.

Northern blot analysis of RNA.
Total cellular RNA was isolated using Trizol reagent (Gibco-BRL), according to the manufacturer's protocols. Total RNA (10 µg) was separated on a 0·8 % agarose/formaldehyde gel and transferred onto a Hybond-N membrane (Amersham). RNA was hybridized to a 32P-labelled {Psi}-specific probe (Shounan et al., 1996) and quantified by phosphorimage analysis. RNA loading was evaluated by probing the same membrane for {beta}-actin or by 18S RNA measurement of ethidium bromide-stained gels before blotting.

Statistical analysis.
Statistical analyses were performed using Student's t-test. Differences were considered significant if P<0·05.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Retrovirus vector titre correlates with GFP expression in retrovirus vector-producing cells
Two means were used to address this potential correlation by assessing the titres of (i) 14 independent PA317/LGL clones and (ii) three sequentially sorted populations of PA317/LGL cells shown to express different levels of GFP. Titres were determined by infection of NIH 3T3 cells and plotted against median GFP fluorescence (Fig. 1). For the 14 producer clones, a correlation (r2=0·46) was found between GFP expression and the titres of virus produced. Pooled, sorted populations were more consistent with a tighter linear relationship between these two parameters (r2=0·91). Four clones were excluded from clonal analysis because when tested for histone acetyltransferase-resistance these four clones were found to be sensitive, showing high toxicity (death). This indicates that these clones had lost or rearranged the thymidine kinase-containing construct. Thus, for cells that do retain an intact construct, GFP expression correlated with virus titre and this served as a means to select high titre producer clones.



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Fig. 1. Relationship between fluorescence and virus titre. GFP expression in PA317/LGL cells was measured by flow cytometry. Virus titre was determined by infection of NIH 3T3 cells and is expressed as virus particle (vp) ml-1. The titre of PA317/LGL clones and sorted pooled populations was plotted against the corresponding median fluorescence seen in virus-producing cells.

 
Expression of the integrated provirus does not depend on cell cycle stage
It has been shown previously that the HIV-1 and HTLV LTRs were activated by G2/M growth arrest (Maio et al., 1995). In addition, the HIV-1 Vpr protein was shown to increase virus expression and infection by arresting host cells at the G2/M phase (Goh et al., 1998). Retrovirus vector-producing PA317/LGL cells (68 % cells were GFP positive) were treated with nocodazole, an agent which prevents cytoskeleton formation, thereby preventing mitosis and arresting cells at the G2/M phase (Zieve et al., 1980; Dolnikov et al., 2000), or aphidicolin, a known DNA polymerase inhibitor that blocks cells at the G1 phase (Huberman, 1981). Comparisons were made of (i) exponentially growing cells, which, relative to the other treated cells, are enriched with S phase cells, (ii) nocodazole-treated cells enriched with G2/M cells and (iii) cells treated with aphidicolin and enriched with G1 cells (Fig. 2A, panels a–c). When mean fluorescence intensity (MFI) in GFP-positive cells was measured, neither nocodazole nor aphidicolin treatment upregulated GFP expression compared to untreated cells (Fig. 2A, panel d). Thus, G2/M or G1 arrest alone did not upregulate retrovirus GFP expression in virus-producing cells. This was also the case for TF-1 cells infected with retrovirus vector produced by PA317/LGL cells (90 % cells were GFP positive) – neither nocodazole-induced G2/M arrest nor aphidicolin-induced partial G1 growth arrest modified MFI for GFP (Fig. 2A, right panels). In addition, aphidicolin did not produce complete G1 growth arrest in TF-1 cells – increased concentrations of aphidicolin induced apoptosis rather than further growth arrest (data not shown). These results were confirmed further by showing that neither nocodazole nor aphidicolin affected retrovirus vector transcription in PA317/LGL cells, as assessed by mRNA quantification (Fig. 2B). Both retrovirus vector and {beta}-actin expression appear to be reduced in aphidicolin- or nocodazole-treated cells (Fig. 2B). Apparently, this was due to non-specific toxicity of these drugs, as reported previously (Holt et al., 1997).



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Fig. 2. (A) Cell cycle analysis and GFP expression in aphidicolin- and nocodazole-treated and untreated cells. Polyclonal virus-producing PA317/LGL cells and human TF-1 cells infected with the retrovirus vector produced by PA317/LGL cells were treated overnight with aphidicolin or nocodazole. GFP expression and cell cycle analysis were performed by flow cytometry. Cell counts are shown on the y-axis and GFP fluorescence is shown on the x-axis. Panels: a, cell cycle analysis of untreated cells; b, cell cycle analysis of nocodazole-treated cells; c, cell cycle analysis of aphidicolin-treated cells; d, GFP expression in untreated (solid line), aphidicolin-treated (dotted line) and nocodazole-treated (solid dotted line) cells; these lines clearly overlap one another. One representative experiment of three is shown. (B) Northern blot analysis of retrovirus transcription in untreated PA317/LGL cells or cells treated with aphidicolin or nocodazole. Total RNA was isolated from cells treated with aphidicolin, nocodazole or untreated cells. A 2·4 kb retrovirus transcript was visualized by probing with a 32P-labelled retrovirus-specific probe. A 2·1 kb {beta}-actin probe was used as a loading control. RNA was quantified by phosphorimage analysis and normalized values for retrovirus expression are shown below.

 
As another means to assess potential cell cycle modulation of provirus gene expression, we next analysed retrovirus vector expression in G1, S and G2/M subpopulations of untreated, exponentially growing vector-producing cells. Cell permeabilization, critical for PI DNA staining in living cells, greatly diminished GFP fluorescence (data not shown). Therefore, we chose to analyse NGFR expression in PA317/NGFR cells and Jurkat T-cells infected with NGFR retrovirus vector (Fig. 3). Normalization was made to the relevant measurements in cells treated with non-specific IgG antibody (PA317/NGFR cells) (Fig. 3A, B) and to the measurements performed in the NGFR-negative subpopulation (Jurkat cells) (Fig. 3C). No significant differences in MFI for NGFR expression were seen in G1 (2 N), G2/M (4 N) and S phase (2–4 N) subpopulations (Fig. 3).



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Fig. 3. Retrovirus NGFR expression in virus-producing cells and in human Jurkat cells infected with virus. Cells were stained with a primary antibody to NGFR or unspecific IgG as a control followed by a secondary FITC-conjugated anti-IgG. Cells were then permeabilized and stained with PI for cell cycle distribution. Two-parameter flow cytometry was performed to analyse NGFR expression and cell cycle. (A) Dot-plot analysis of PA317/NGFR cells stained by IgG. (B) Dot-plot analysis of PA317/NGFR cells stained with NGFR-specific antibody. (C) Dot-plot analysis of Jurkat/NGFR cells stained with NGFR-specific antibody. NGFR expression is shown on the x-axis and PI staining of DNA is shown on the y-axis. A line indicating the chosen cut-off value to distinguish NGFR-positive cells is shown.

 
S phase-enriched cell cultures produce more virus
Experiments to this point addressed provirus expression. We next asked whether cell cycle modulation could impact on retrovirus vector production steps subsequent to provirus expression. Retrovirus vector-producing cells were plated at 2·5x105 cells ml-1 and VCM was harvested at later time points and assessed by infecting target NIH 3T3 cells. Total and ‘normalized’ bulk gene transfer was assessed, the latter by normalizing to producer cell counts and production time. Results showed that while the overall proportion of transduced cells increased with time of VCM production (Fig. 4A, panel a), the proportion of transduced cells, when normalized to producer cell counts and production time, decreased with time of culture (Fig. 4A, panel b). Concomitant with this ‘normalized’ decrease in virus titre is a decline in the proportion of S phase cells from 56 % at 3·25 h to 28 % at 23 h (data not shown). These results indicate that S phase cells produce virions more efficiently per cell per unit time.



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Fig. 4. Virus titre correlates with the proportion of S phase cells in producer cell culture. (A) PA317/LGL cells were plated at 2·5x105 cells ml-1 and VCM was harvested at time points of 3·25, 4·5, 8 and 23 h later. These virus harvests were used to infect target NIH 3T3 cells (panel a). Differences were significant (P<0·05) between points 1 and 2, 1 and 5, and 1 and 4. The percentage of transduced cells was normalized to producer cell counts at the time of harvest and expressed as production per unit time (panel b). (B) Confluent PA317/LGL cells were split 1 : 5, 2 : 5, 3 : 5 and 4 : 5 to approximate 20, 40, 60 and 80 % confluency; virus was harvested 3 h later and used to infect NIH 3T3 cells. Cell cycle analysis of producer cells was performed at the time of virus harvest. Transduction efficiency was normalized to producer cell counts. Differences were significant (P<0·05) between points 1 and 3, 1 and 4, 2 and 4, and 3 and 4. (C) PA317/LGL cells were incubated with 0·1 % serum to induce G1 growth arrest. At 96 h, 10 % serum was added to release arrest. VCM was collected 2·5 h following release from growth arrest. Fresh medium was added and VCM was collected 2·5 h later. This was repeated once again. VCM was used to infect NIH 3T3 cells. Cell cycle analysis of producer cells was performed at the time of virus harvest. Transduction efficiency was normalized to producer cell counts and expressed as production per 1 h. Differences were significant (P<0·05) between all three time points.

 
We used two methods to examine this further. In the first method, we seeded PA317/LGL cells at different cell densities by splitting confluent cultures 1 : 5, 2 : 5, 3 : 5 and 4 : 5, thereby approximating 20, 40, 60 and 80 % confluency. After 3 h, producer cells and VCM were harvested. Virus titre and cell cycle analyses were then performed. A correlation was found between the percentage of S phase cells and the ‘normalized’ titre (Fig. 4B). In the second method, we arrested virus-producing cells at the G1 phase by serum depletion (0·1 % serum) and then released them by seeding at relatively low cell density (1 to 2x105 cells ml-1) into medium containing 10 % serum. Virus titre and cell cycle analyses were performed 2·5, 5 and 7·5 h later. Again, a positive correlation was found between the proportion of S phase cells and virus titre (Fig. 4C).

Parameters of retrovirus vector harvest
We next asked how best to optimize retrovirus vector harvest by analysing certain variables: percentage S phase cells and virus vector decay. Retrovirus vectors are often produced by overnight (16 h) culture. We assessed the efficacy of such a 16 h collection schedule (I) compared to a single 8 h collection (II), during which time the percentage of cells in the S phase declined from approximately 60 % to approximately 20 %, a second 8 h collection (III) when producer cells were in confluent phase (approximately 20 % of the cells were in the S phase within the whole 8 h period), and the addition of an 8 h collection of VCM to the initiation of another 8 h collection (IV). In all cases, cells were seeded at approximately 60 % confluency. There are several points to note from the results. Firstly, VCM produced during the first 8 h (II) and the second 8 h (III) periods of a 16 h retrovirus vector preparation (I) both infected a similar percentage of NIH 3T3 cells (Fig. 5A). It is relevant that collection II was produced by cultures with the higher percentage of S phase cells and lower total cell number compared to collection II. Secondly, these collections were similar to 8+8 h split retrovirus vector harvest (IV), due presumably to the effect of virus decay (Fig. 5B). Thirdly, vector produced during the 16 h collection (I) infected a lower percentage of NIH 3T3 cells compared to collections II, III or IV (Fig. 5B). The difference between a 16 h collection (I) and two sequential 8 h collections (IV) points to the effect of the higher percentage of S phase cells in collection IV, while the difference between collections I and II and I and III points presumably to differences in vector decay. Collection IV did not produce a higher titre compared to collections II and III, also due presumably to vector decay.



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Fig. 5. An 8 h virus harvest is more efficient in cell transduction compared to a 16 h harvest. (A) Virus-producing cells were plated at 60 % confluency and virus was harvested at 16 h (I), 8 h (II) or medium was replaced after the first 8 h with fresh medium to collect a second 8 h harvest (III). In addition, the first 8 h harvest was placed on freshly seeded producer cells and incubated for an additional 8 h to produce an 8+8 h split harvest (IV). VCMs I, II, III and IV were used to infect NIH 3T3 cells (overnight incubation) and transduction was measured 48 h following infection. One representative experiment is shown. Infections were performed in triplicate. Differences were significant (P<0·05) only between I and II, and III and IV (*). (B) Virus titre half-life. Virus titre was measured over time during incubation at 37 and 32 °C. A line of exponential decay was fitted to data points and had high r2 values over 0·9. Virus had half-lives of 5·5 h at 37 °C and 16–20 h at 32 °C.

 
In the next series of experiments, retrovirus vector half-life was calculated by incubating VCM for varying lengths of time at 37 or 32 °C. Virus titre (measured on NIH 3T3 cells) was found to drop with time and retrovirus vector half-life was calculated by drawing exponential lines of best fit (Fig. 5C). For both packaging cell lines analysed (PA317 and PG13), retrovirus vectors had half-lives of approximately 5·5 h at 37 °C and 20 h at 32 °C.

Target cell transduction: synchronous progression of TF-1 cells through S to G2/M increases retrovirus transduction
For MoMLV and similar retroviruses, entry into mitosis is critical for efficient provirus DNA integration (Roe et al., 1993; Lewis & Emerman, 1994). G1 cells often make up the majority of cells in a population and even for rapidly cycling cells like NIH 3T3 it takes more than 11 h for early G1 cells to reach mitosis (Karn et al., 1989). This is important in terms of retrovirus transduction because the half-life of pre-integration complexes has been shown to be in the range of 5·5 to 7·5 h (Andreadis et al., 1997). The challenge for maximizing transduction thus becomes how to ensure maximum exposure of mitotic cells to transducing retrovirus vector.

We sought to analyse whether synchronization of cells close to mitosis and prior to infection may increase the number of cells susceptible to transduction. Human leukaemic TF-1 cells with a doubling time of approximately 36 h (our unpublished data) were incubated with aphidicolin overnight to arrest the cells at the G1/S phase (Fig. 2A, right panels). Cells were then released from growth arrest and exposed briefly (3 h) to vector at different time points following release. The highest levels of infection were seen when cells were exposed to the virus 24 h after the release from the aphidicolin block, slightly decreased at 27 and 29 h (Fig. 6A). However, it was clear that there was a significant difference between the synchronized culture at 24 h and the unsynchronized population at the same time point (Fig. 6A). TF-1 cells began to enter S phase 6 h after release from growth arrest and a majority of cells accumulated in S to G2/M phases at 25 to 29 h after release from growth arrest (Fig. 6B). This effect is not due to multiplicity of infection, as the target cell number remained relatively constant over this protocol. As mentioned previously, aphidicolin did not produce complete synchronization of TF-1 cells at the G1 phase (Fig. 2A, panel c). It may be that more efficient synchronization would increase further the degree of transduction of TF-1 cells. This effect may have been due to post-integration effects but this possibility is less likely due to the lack of effect in NIH 3T3 cells.



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Fig. 6. Synchronization of the cells by aphidicolin before exposure to the virus increases infection efficiency of TF-1 but not NIH 3T3 cells. Aphidicolin was added to the exponentially growing TF-1 (A) or NIH 3T3 (C) cells to synchronize at the G1/S phase. At 24 h later, cells were released from growth arrest and virus was added at several time points for 3 h. Cells were infected at an m.o.i. of 10. GFP expression was measured 48 h following infection. Measurements were done in triplicate. (B) Percentage of TF-1 cells in the S phase at various time points following release from G1/S growth arrest. Measurements of cell cycle were done in duplicate and averages were plotted. Results of one representative experiment are shown. Differences between unsynchronized cells and cells that were synchronized by aphidicolin were considered as significant (P<0·05). The differences between 24 and 27 h samples were not significant.

 
In contrast, aphidicolin-mediated synchronization did not affect infection efficiency of rapidly cycling NIH 3T3 cells (doubling time 14 h) (Karn et al., 1989). As for TF-1 cells, NIH 3T3 cells were incubated with aphidicolin overnight and then released from aphidicolin-mediated G1/S growth arrest. It is relevant that aphidicolin-mediated synchronization of NIH 3T3 cells at the G1/S phase was more efficient than for TF-1 cells and cell cycle pattern was similar to NIH 3T3-based PA317 cells (Fig. 2A, panel c, left panel). Cells were exposed briefly (2 h) to the retrovirus vector 2, 4 and 6 h following release from growth arrest. The level of infection was found to be similar in control and synchronized cells (Fig. 6C). These shorter times were chosen as it takes only 6·5 h for NIH 3T3 cells to progress from G1 through S to G2/M (data not shown).

We next examined the ability of the recombinant fibronectin fragment CH296, RetroNectin, shown previously to improve retrovirus transduction by co-localization of virus particles and target cells (Moritz et al., 1996), to improve retrovirus transduction of aphidicolin-synchronized TF-1 cells. As expected, RetroNectin, the present method of choice, increased retrovirus transduction of untreated TF-1 cells. This was to a similar level as that of aphidicolin synchronization: proportion of infected RetroNectin-treated cells was 17·7±0·8 % compared to 8·0±0·5 % in untreated cells and 14·4±0·6 % in cells treated with aphidicolin. RetroNectin did not increase transduction of aphidicolin-treated cells further (14·8±1·0 %).

We next attempted to synchronize TF-1 cells using nocodazole. Nocodazole-treated cells accumulated at the G2/M point (Fig. 2A, panel b). These cells were subjected to the virus either 3 h before or 3 h after release from growth arrest. The time period of incubation with virus was 3 h. We found that cells synchronized by nocodazole were infected more efficiently than untreated cells and that 3 h prior was better that 3 h post release from growth arrest (Fig. 7). Retrovirus transduction of nocodazole-treated cells exposed to the virus 3 h prior to release from growth arrest and incubated with virus for only 3 h was similar to that produced by 24 h of incubation of non-synchronized cells. It is relevant that a 24 h incubation of synchronized cells increased the percentage of transduced cells further when compared to a 24 h incubation of non-synchronized cells (Fig. 7). It is relevant that, in addition to the increased proportion of transduced cells, the MFI was significantly higher in nocodazole-treated cells (Fig. 7). This may be due to increased provirus copy number in transduced cells when transduction level is high. Thus, nocodazole treatment, which produced efficient synchronization of TF-1 cells prior to mitosis and reduced asynchronous progression of TF-1 cells through mitosis during their exposure to the retrovirus vector, significantly increased the level of gene expression. In addition, this treatment maximizes retrovirus gene transfer within a shorter period of time.



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Fig. 7. Synchronization of TF-1 cells at the G2/M phase by nocodazole increases transduction efficiency. Cells were incubated with nocodazole overnight. Cells were with virus 3 h before or 3 h after release from the block. The time period of incubation was 3 or 24 h. Results of one representative experiment are shown.

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Here we used markers to determine means to analyse the effect of cell cycle on retrovirus vector production. We first used these markers to identify high-titre packaging cell lines and showed that retrovirus vector titre correlates with GFP expression in vector-producing cells. Thus, rapid screening for the most effective retrovirus vector producers can be performed based on GFP expression in producer cells. GFP expression is thus a measure of virus expression. However, this correlation between GFP expression and virus production should always be a first step with secondary screens used to identify strong virus producers.

Retrovirus replication occurs through a DNA intermediate and provirus DNA transcription appears to be an important step in virus production. Given that MoMLV-driven GFP expression in the retrovirus vector-producing cells correlated with the virus titre produced by these cells, upregulation of retrovirus vector expression may improve retrovirus vector titre further. The HTLV-1 and HIV-1 LTRs were shown previously to be upregulated in cells arrested at the G2/M phase (Maio et al., 1995). In addition, the HIV-1 Vpr protein was shown to increase virus expression by arresting cells at the G2/M phase (Goh et al., 1998). This prompted us to test whether G2/M growth arrest activated MoMLV LTR-driven transcription in retrovirus vector-producing cells similar to these other promoters. We first tested nocodazole, which arrests cells at the G2/M phase and found that retrovirus expression of GFP was not upregulated by this approach. Confirming these findings, Northern blot analysis demonstrated similar levels of retrovirus vector-specific transcript in untreated, nocodazole- or aphidicolin-treated producer cells. Thus, G2/M growth arrest alone is not sufficient to upregulate MoMLV LTR activity, at least in these cells. In addition, similar amounts of reporter gene (human truncated NGFR) expression were seen in the G1, S and G2/M subpopulations of untreated, exponentially growing cells. These results are consistent with the results of MoMLV LTR-driven expression of large T antigen in immortalized astrocyte lines showing that large T antigen levels did not change during exponential growth neither as a function of the G1 fraction nor of increasing cell density (Frisa & Jacobberger, 2002).

The promoter/enhancer elements apparently necessary for 2-aminopurine-induced activation of retrovirus expression have not been identified precisely. However, at least two elements seem to be required: two enhancer sequences and the Sp1 site of the HIV LTR (Maio et al., 1995; Agarwal et al., 1998). In addition, it has been shown that activation of basal transcription of some cellular promoters at the G2/M point of the cell cycle is mediated through Sp1 by the transient de-repression of transcription inhibited by the CDE/CHR box at the G1 phase of the cell cycle (Dando et al., 2001). All of these elements are present in the MoMLV LTR. Apparently, some unidentified cis-acting inhibitory elements may prevent activation of similar sequences in the MoMLV LTR.

In addition, the SV40 promoter, shown previously to be upregulated by G2/M growth arrest in human U937 cells (Maio et al., 1995), was not affected by G2/M growth arrest in murine NIH 3T3 cells (data not shown). Apparently, NIH 3T3 cells as well as NIH 3T3-based retrovirus vector-producing cells lack transcription factors that are upregulated by G2/M arrest in human U937 cells (Maio et al., 1995). MoMLV LTR activity is examined currently in human retrovirus vector-producing cells in association with cell cycle. Increased MoMLV LTR activity is likely to be beneficial, particularly for transient retrovirus vector production, since upregulation of HIV-1 LTR by G2/M growth arrest was shown to be particularly efficient in transient (episomal) expression systems (Maio et al., 1995).

We next showed that although MoMLV LTR GFP expression appears to be similar at all phases of the cell cycle, the amounts of retrovirus vector produced by exponentially growing cells are higher compared to confluent cultures. There was a direct correlation with the percentage of cells in the S phase. This was demonstrated in a number of experiments in which the percentage of S phase producer cells was modulated. These included (i) a decrease in virus production over time related to the decrease in the percentage of S phase cells, (ii) a positive relationship between virus titre and the percentage of S phase cells derived by seeding at different cell densities and (iii) a positive relationship between virus titre and the percentage of S phase cells in serum withdrawal-synchronized cell culture. Thus, the experiments establish a direct relationship between the proportion of S phase cells in producer cells culture and virus titre.

We addressed further the effects of cell cycle and time of harvest on virus titre. We demonstrated that an 8 h harvest was superior to a 16 h harvest and this is due presumably to the increased percentage of S phase cells and low virus decay in the 8 h collection compared to the 16 h collection. In addition, two sequential 8 h split harvests produced virus titres similar to the first or second 8 h period of the total 16 h preparation. We also demonstrated that two sequential 8 h harvests could be conducted on the same cells yielding similar titres. We showed that the half-life of retrovirus vectors produced by two murine packaging cell lines PA317 and PG13 cells at 37 °C was 5·5–6 h and increased up to 20 h at 32 °C. These results are consistent with the half-life estimations determined for the human packaging FLYRD18/LNC-hB7 cells (6·9 h at 37 °C and 11 h at 32 °C) (McTaggart & Al-Rubeai, 2000).

Based on our retrovirus vector half-life estimations, it appears that virus decay contributes significantly to the second 8 h harvest. Thus, it would appear that the second 8 h harvest could be performed better at 32 °C when virus decay is less. Interestingly, it was shown recently that while optimal virus harvests can be obtained when culturing certain packaging cells (PA317) at 32 °C, this does not, however, apply for other packaging cells (PG13) (Reeves et al., 2000).

Our results are consistent with recent data showing that time of retrovirus vector production by the GP+AM12 packaging cell line appeared to have the greatest impact on gene transfer, peaking at 6 h and decreasing two- to threefold at longer time points (Dando et al., 2001). In the present experiments, it is likely that the contribution of S phase cells to retrovirus vector production and low retrovirus vector decay may account for the increased virus titres produced by the 8 h harvests by the PA317 and PG13 packaging cell lines. Our results, however, are not totally consistent with the results of Reeves and co-workers, showing that with the vectors used, PA317 and PG13 cells, yielded optimal titres after 24 h of culture (Reeves et al., 2000). This apparent discrepancy could be due to the differences in cultures conditions: in our experiments, producer cells were grown in tissue culture flasks, while those of Reeves and co-workers were grown in roller bottles and were used where confluency was reached at 24 h rather than 8 h.

It was shown previously that the low stability of virus particles appears to be one of the intrinsic limitations affecting the efficiency of retrovirus infection and thus efficient retrovirus transduction requires a rapid time period, as the effective time of exposure of target cells to the retrovirus vector would be limited (Andreadis et al., 1997). Cell cycle analysis of retrovirus vector gene expression revealed that retrovirus vector expression was first detected exclusively in the late G1 and early S phases of the cell cycle (Sladek & Jacobberger, 1998). These results suggest that, at the time of infection, cells in certain phases of the cell cycle were more susceptible to infection than cells in other phases.

In addition, it was shown that in actively dividing cells, the transport of the pre-integration complex to the nucleoplasm requires the breakdown of the nuclear membrane, which occurs during mitosis (Miller et al., 1990; Roe et al., 1993). Therefore, it is critical for target cells to pass through mitosis within 5·5–7·5 h after exposure to retrovirus vector, since, during this time, a significant clearance of retrovirus vector occurs inside the cell (a 5·5–7·5 h half-life was established previously for pre-integrated intracellular virus complexes) (Andreadis et al., 1997).

Primitive HSCs appear to be one of the most appropriate targets for gene therapy, since gene transfer in such cells may produce long-term effects. As HSCs have a low mitotic index, cytokines are used for induction of these cells into the cell cycle (Williams et al., 1997). It was reported that different subpopulations of human CD34+ cells appear to be engaged in and progress through the cell cycle asynchronously with an average doubling time of 72 h (Traycoff et al., 1998). It is relevant that primitive murine haematopoietic cells when stimulated to enter the cell cycle have a G1 phase of about 18 h (Traycoff et al., 1998). Thus, by the time these cells reach mitosis, they will be exposed to significantly reduced concentrations of virus. In this context, it is not surprising that the percentage of retrovirus vector gene transfer into CD34+ cells was found to be significantly lower than the percentage of cells engaged in the cell cycle (Williams et al., 1997; Traycoff et al., 1998).

We reasoned that synchronous progression through the cell cycle may increase the efficiency of infection. To assess this, human CD34+ TF-1 cells with a doubling time of approximately 36 h were examined. Two synchronizing procedures were examined. Firstly, cells were blocked at the G1/S phase with aphidicolin and then released from this block to progress synchronously through S phase and mitosis. Cells were exposed to the retrovirus vector at different time points following release from the aphidicolin block. The most efficient time point for infection was found to be 24–25 h following release from the block when cells are progressing through S to G2/M. TF-1 cells began to progress from the G1 to the S phase 6 h after release from aphidicolin (Fig. 7). Apparently, this 6 h latency was critical for TF-1 cells to recover from aphidicolin treatment. In contrast, cell synchronization did not affect the susceptibility of rapidly cycling NIH 3T3 cells to infection, due presumably to the rapid cell cycle. In addition, we demonstrated that the addition of RetroNectin, which significantly improves retrovirus vector transduction, did not increase further the proportion of infected aphidicolin-synchronized TF-1 cells.

The effect of synchronization on the efficiency of infection was even more significant when TF-1 cells were arrested at the G2/M phase by nocodazole. Nocodazole-treated cells subjected to the retrovirus vector 3 h before or 3 h after release from the nocodazole block were infected more efficiently compared to untreated cells: both increased the proportion of transduced cells as well as MFI. In addition, the level of retrovirus transduction of nocodazole-synchronized cells exposed to the virus 3 h before release from growth arrest and incubated with the virus for just 3 h was similar to that produced by 24 h of infection of non-synchronized cells. A 24 h infection period of the synchronized cells further increased transduction efficiency. We have shown that the optimal steps for virus harvest and transduction are (i) an 8 h harvest from the producer cells to optimize virus titre and (ii) nocodazole synchronization of TF-1 target cells at the G2/M phase to optimize susceptibility to transduction. The practical significance of increased transduction rates following cell synchronization is that it allows the overall time of infection to be reduced: a short exposure time may lead to infection levels that would otherwise require more extensive exposure, such as multiple exposures. Multiple exposures may not be possible for primary cells, such as HSCs, as increasing the length of culture drives differentiation; prolonged ex vivo culture has been shown to reduce stem cell primitiveness, increase apoptosis and decreased engraftment (Traycoff et al., 1998; Xu & Reems, 2001). Thus, synchronization of primitive haematopoietic cells prior to infection may act to reduce the time period of overall ex vivo culture. In this regard, we have found that the titration of nocodazole dose/time of treatment is difficult for human haematopoietic progenitor cells given the heterogeneity of the CD34 cell population, particularly in relation to cell division. We believe that this approach will require the use of a dye, such as carboxyfluorescein diacetate succinimidyl ester, for marking of the cells to monitor the degree of synchronization obtained. It is relevant that human haematopoietic CD34+ TF-1 cells, which were used to assess transduction in this study, are in many respects reminiscent of primary haematopoietic progenitor cells and we have found that virus titres assessed using TF-1 cells correlate with the transduction of primary CD34+ cells.


   ACKNOWLEDGEMENTS
 
The authors thank Dr Sylvie Shen for technical assistance and Toby Passioura for helpful advice and discussion. This work was supported by NHMRC grant number 113821. Dr Geoff Symonds was an NHMRC Principal Research Fellow during the execution of this work. We gratefully acknowledge facility space at Johnson and Johnson Research Laboratories.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 15 January 2003; accepted 12 June 2003.



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