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Stem Cells 2003;21:472-480 www.StemCells.com
© 2003 AlphaMed Press

Highly Efficient Lentiviral Gene Transfer in CD34+ and CD34+/38-/lin- Cells from Mobilized Peripheral Blood after Cytokine Prestimulation

Fabien Géronimi, Emmanuel Richard, Isabelle Redonnet-Vernhet, Isabelle Lamrissi-Garcia, Magalie Lalanne, Cécile Ged, François Moreau-Gaudry, Hubert de Verneuil

Federative Research Institute 66, Bordeaux, France

Key Words. Lentiviral vector • Gene transfer • CD34+ • CD34+/38-/lin- • Mobilized peripheral blood • Prestimulation

Hubert de Verneuil, MD., PhD., INSERM E0217, Université Victor Segalen Bordeaux 2, 146 rue Léo Saignat, 33076-Bordeaux Cedex, France. Telephone: 33-5-57-57-13-70; Fax: 33-5-56-98-33-48; e-mail: verneuil{at}pmtg.u-bordeaux2.fr


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Because mobilized peripheral blood (mPB) represents an attractive source of cells for gene therapy, we investigated lentiviral gene transfer in CD34+ cells and the stem/progenitor-cell-enriched CD34+/38-/lin- cell subset isolated from mPB. In this study, we used an optimized third-generation self-inactivating lentiviral vector containing both the central polypurine tract and the woodchuck hepatitis posttranscriptional regulatory element sequences and encoding enhanced green fluorescent protein (EGFP) under the control of the elongation factor l{alpha} promoter. This lentivector was first used to compare multiplicity of infection (MOI)-dependent gene transfer efficiency in cord blood (CB) versus mPB CD34+-derived cells, colony-forming cells (CFCs), and long-term culture-initiating cells (LTC-ICs). Results showed a difference in the percentage of transduced cells particularly significant at low MOIs. A plateau was reached where 15% and 25% of CB and mPB cells, respectively, remained refractory to lentiviral trans-duction. Effects of a cytokine prestimulation period (18 hours) with interleukin-3, stem cell factor, Flt-3 ligand, and thrombopoietin were then analyzed in total cells, CFCs, and LTC-ICs derived from mPB CD34+ cells. Transduction levels in those conditions demonstrated a two- and fourfold increase in CFCs and LTC-ICs, respectively, compared with unstimulated (<3 hours) control cells. Moreover, using the same transduction protocol, we were able to efficiently transduce CD34+/38-/lin- cells isolated from mPB, with up to >85% of colonies derived from LTC-ICs expressing EGFP and gene transfer levels remaining stable for 10 weeks in liquid culture. We therefore demonstrate a highly efficient gene transfer in this therapeutically relevant target cell population.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
For nearly two decades, gene therapy has been a major field of investigation for the treatment of a variety of hematological diseases including congenital disorders, cancer, and HIV infection [1–3]. Long-term genetic correction of hematolymphoid cells relies on the efficient transfer of therapeutic genes into human hematopoietic stem cells (HSCs), the only population of cells capable of life-long self-renewal and maturation to the various blood cell types. To achieve this goal, Moloney murine leukemia virus (MMLV)-derived vectors have been widely used, and transduction protocols have been considerably optimized, especially with the use of early-acting cytokines and a recombinant fibronectin peptide CH-296 [4–6]. These vectors have proven high transduction efficiencies, stable gene expression, lack of immuno-genicity, and ease of vector production, relative to other types of vectors [7]. However, despite reported efficient long-term gene transfer in murine and human progenitor cells, a number of preclinical studies involving transplantation of gene-modified cells in large animals and humans has demonstrated the absence of durably detected high levels of transduced cells [8–10]. One of the major explanations is the requirement of cell division for nuclear entry of the MMLV genome [11]. Unfortunately, in their native state, most long-term HSCs are quiescent, and attempts to stimulate these cells into the cell cycle in vitro have impaired their in vivo long-term repopulating capacities [12, 13]. The difficulty of transducing HSCs is emphasized when working on cells isolated from mobilized peripheral blood (mPB) instead of cord blood (CB) or bone marrow (BM) [14]. These different sources of cells are attractive targets for many gene therapy protocols, but MMLV-based transduction strategies are insufficient for diseases where there is no selective advantage of corrected cells.

A major improvement has come in the past few years with the increasing use of HIV-based vectors. These lentivectors have been extensively modified and now provide safe and efficient gene transfer vehicles for HSCs. A number of investigators have reported efficient transduction of nondividing quiescent or cell-cycle-arrested cells from different human tissues [15–20]. However, recent findings demonstrate that, although CD34+ cells in G0 can be transduced, gene transfer is much more efficient in CD34+ cells in G1or S/G2/M phase [21].

In this study, we report differences in lentiviral gene transfer efficiencies in mPB versus CB CD34+ cells. While the transduction of mPB long-term culture-initiating cells (LTC-ICs) in a nonprestimulated (or very shortly prestimulated) protocol is low, a prestimulation of cells with early-acting cytokines allowed very high transduction levels (over 80%) in total and progenitor cells and a 60% gene transfer rate in LTC-ICs derived from mPB cells. Moreover, using the same optimized protocol, we were able to transduce CD34+/38-/lin- cells isolated from mPB at very high levels, with >85% of LTC-ICs expressing the trans-gene and with a sustained percentage of transduced cells in long-term culture.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Lentiviral Vector Constructs
Trip-{Delta}U3-EF1{alpha} (TEE) vector was a generous gift of P. Charneau from the Institut Pasteur, Paris, France. Woodchuck hepatitis posttranscriptional regulatory element (WPRE) was cloned between the Xhol and the Kpnl sites of TEE vector to obtain TEEW (Fig. 1AGo).



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  		Figure 1. Lentiviral gene transfer in CD34+ cells from CB or mPB. A) Schematic representation of lentiviral vector constructs. SD = splice donor; SA = splice acceptor sites; RRE = rev responsive element; cPPT = central polypurine tract; CTS = central termination sequence; EF1{alpha} = human elongation factor 1{alpha} promoter; WPRE = woodchuck hepatitis posttranscriptional regulatory element; AU3 = U3 region deleted of TATA box and enhancer sequences. TEE vector was a kind gift of P. Charneau (Institut Pasteur, Paris, France). B) CD34+ cells from CB and mPB were transduced in a short protocol with <3 hours of cytokine exposure on fibronectin CH-296-coated plates. The percentages of EGFP-expressing cells were determined by flow cytometry, 3 days after the second transduction, on the whole cell population, for various MOI (values are final MOI: MOI 100 means two transductions at an MOI of 50). C) Percentage of CFC-derived colonies expressing EGFP, for final MOIs of 100 and 400. D) Percentage of LTC-IC-derived colonies expressing EGFP. Values are means and standard deviations from three to five independent experiments (but only two for CB LTC-ICs, at both MOI). *Significant difference (p < 0.05) versus mPB cells transduced at the same MOI using the Mann-Whitney test. = mPB cells; {blacksquare} = CB cells.

 
Virus Production
Human kidney 293T cells (5 x 106) were plated on 10-cm plates, transfected the following day with the packaging construct pCMV{Delta}8.91 (10µg), a vesicular stomatitis virus glycoprotein (VSV-G) envelope plasmid pMD.G (2 µg), and a vector construct (10µg) by calcium phosphate DNA precipitation, and induced with 10 mM sodium butyrate (Sigma; St. Louis, MO; http://www.sigmaaldrich.com). Twenty-four and 48 hours later, viral supernatants were collected in serum-free X-VIVO 10 medium and filtered through 022-µm filters. Viral supernatants were concentrated by centrifugation at 35,000 g for 4 hours at 4°C, immediately or after cryopreservation at -80°C. Infectious liters were determined by transducing 293T cells with serial dilutions of viral supernatant. EGFP expression was quantified 4 days later by flow cytometry. Lentiviral vector preparations were tested for the presence of replication-competent lentivirus (RCL) as described elsewhere [20] and were found to be free of RCL.

Isolation of CD34+ and CD34+/38-/lin- Cells and Culture Conditions
Mononuclear cells from fresh umbilical CB were obtained by centrifugation on Ficoll-Paque Plus® (Amersham Pharmacia Biotech; Orsay, France; http://www.apbiotech.com). Cells were washed three times with phosphate-buffered saline (PBS) containing 2 mM EDTA. CD34+ cells were isolated by two rounds of positive selection using the Mini/Midi MACS system and CD34 progenitor cell isolation kit (Miltenyi Biotec; Paris, France; http://www.miltenyibiotec.com). Mobilized peripheral blood CD34+ cells were obtained from apheresis preparations of patients with myeloma. All cells were cryopreserved until use. The purity of CD34+ cells was assessed by flow cytometry and was found to be >90% at the initiation of the culture. CD34+/38-/lin- cells were isolated from freshly thawed mPB CD34+ cells using the StemSepTM’ system with the primitive hematopoietic progenitor cocktail (Stem Cell Technologies France; Meylan, France; http://www.stemcell.com). This fraction accounted for 0.1%–1% of the original CD34+ population. CD34+ and CD34+/38-/lin- cells were grown in suspension cultures initiated at 2.5 x 105 cells/ml in RM-B00 medium (MABIO-International Laboratories; Tourcoing, France; http://www.mabio.net) containing the following human recombinant cytokines (hereafter named RM+CK): 20 ng/ml interleukin (IL)-3, 100 ng/ml stem cell factor (SCF), 100 ng/ml Flt3-ligand (FL), and 100 ng/ml thrombopoietin (TPO); and supplemented with 100 U/ml penicillin-streptomycin at 37°C in a humidified atmosphere containing 5% CO2.

Transduction of CD34+ and CD34+/38-/lin- Cells
CD34+ cells were nonprestimulated (<3 hours cytokine exposure) or prestimulated for 18 hours prior to viral exposure in RM+CK medium. In the first transduction, 3 x 105 cells were transduced twice at 24-hour intervals, with the TEE vector at different MOI in 600 ml of RM+CK medium, and when indicated, in wells coated with fibronectin fragment CH-296 (RetronectinTM, Takara Shuzo Ltd.; Otsu, Japan; http://www.takara-bio.co.jp) at 10 µg/cm2. The second transduction was carried out by replacing the medium with new viral supernatant and fresh medium.

CD34+/38-/lin- cells were prestimulated 18 hours prior to viral exposure in RM+CK medium. Twice, at 24-hour intervals, 5 x 104 to 1 x 105 cells were transduced with the TEEW vector at a final MOI of 100 (2 x 50) in a minimal volume (200–400 µmol/l) of RM+CK medium supplemented with 50 µmol/l deoxynucleotide triphosphates (dNTPs). The second transduction was carried out by adding equal volumes of viral supernatant and fresh medium with dNTPs to the cells. CD34+- and CD34+/38-/lin--derived cells were harvested 72 hours after the second infection for analysis, except for clonogenic assays, which were initiated 24 hours after the second infection.

Clonogenic Assays
Cells were plated in duplicate in 35-mm tissue culture dishes at 200–3,000 cells/ml, with 1 ml of DM-MSO2 of methylcellulose medium (MABIO-International Laboratories) and cultured at 37°C in a humidified atmosphere containing 5% CO2. Total and green fluorescent colony-forming cells (CFCs) were counted after 14 days using an inverted fluorescent microscope (Olympus IX-50; Merck Eurolab; Lyon, France; http://www.vwr.com). For LTC-IC assay, 3 x 103 to 3 x 104 cells were seeded in 24-well plates containing pre-established adherent stromal cell layers (MS-5 cells) and 1 ml of long-term culture medium (MyeloCult H5100; Stem Cell Technologies) supplemented with 10 ng/ml SCF and 5 ng/ml FL, without hydrocortisone. Cells were incubated for 5 weeks at 37°C in a humidified atmosphere containing 5% CO2, with weekly half-medium changes. After 5 weeks of culture, adherent and nonadherent cells were harvested, pooled, and then plated in methylcellulose medium for CFC assay, as described above. Cells recovered from weekly semi-depopulations were analyzed by cytofluorimetry for EGFP fluorescence. For CD34+/38-/lin- cells, long-term cultures were prolonged up to 10 weeks by reseeding week-5 cells on newly established stromal cell layers. Culture conditions remained as described above.

Flow Cytometry
Flow cytometric analysis was performed on a FACSCalibur (BD Biosciences; Le Pont de Claix, France; http://www.bdbiosciences.com) using Cell Quest Pro software. Cells were assessed using mouse anti-human CD34 antibody conjugated to R-phycoerythrin-cyanin 5.1 (PC-5); isotype control was mouse IgG conjugated to PC-5 (Beckman Coulter France Villepinte; Roissy CDG, France; http://www.beckman.com). EGFP fluorescence was detected using detector channel FL-1. During quadrant analysis, coordinates were set to locate >99% of isotype events in the lower left quadrant.

Detection of Provirus Integration
High-molecular-weight DNA was extracted from transduced and untransduced CD34+ cells and analyzed by Southern blot hybridization. A 10-µg amount of DNA was digested with EcoRI and KpnI, separated on a 0.8% agarose gel, and transferred onto nylon membrane (Hybond N+; Amersham). Filters were hybridized with WPRE sequence that was labeled to high specific activity with [32P]-2'-deoxy-cytidine 5'-triphosphate sodium salt by random priming (Rediprime; Amersham). Known amounts of digested plasmid DNA (TEEW plasmid) were mixed with genomic DNA from untransduced cells and run on the same gel. The WPRE probe recognized a 1.8-kb band from the TEEW plasmid in DNA from transduced cells and mixed samples (plasmid + untransduced cells). The vector copy number was estimated by comparison of the amount of radioactivity (Phosphor Imager; Molecular Dynamics; Piscataway, NJ; http://www.amershambiosciences.com). One human cell contains 6 pg of DNA; the radioactivity obtained with 16.7 pg of TEEW plasmid corresponds to the presence of 1 copy per human genome, when 10 µg of genomic DNA is analyzed in the blot.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Comparison of MOI-Dependent Transduction Efficiency in CB Versus mPB CD34+Cells
MOI-dependent lentiviral transduction of CB and mPB CD34+ cells was first studied in total and CD34+ cells and in CFC- and LTC-IC-derived colonies using the TEE vector (Fig. 1AGo). Figure 1BGo shows that CB cells were efficiently transduced whatever the MOI, while mPB cells in the same conditions demonstrated lower gene transfer rates with significant differences at MOI of 20 and 50 (76.0% versus 46.6% and 81.9% versus 58.6% for CB and mPB cells, respectively). Results for cells still expressing CD34+ cells after 6 days of culture were similar to what was observed in the bulk population (data not shown). Concerning CFC-derived colonies, differences between CB and mPB were as observed for total cells, with colonies derived from mPB cells being less efficiently transduced (Fig. 1CGo). When looking at LTC-IC-derived colonies, dramatic differences were observed between CB and mPB, with 61.7% ± 9.7% and 15% ± 2.0% of EGFP+ cells, respectively for an MOI of 100, and 77.0% ± 9.9% and 23.7% ± 7.5% for an MOI of 400 (Fig. 1DGo).

We also monitored the percentages of EGFP+ cells in long-term liquid cultures. While the proportion of EGFP+ cells in transduced CB cells was maintained throughout the 5 weeks of culture, the percentage of transduction in mPB cells decreased, mainly during the first week of culture and approximately to the same extent regardless of the MOI (Fig. 2Go). This decrease is probably partly due to a persistent pseudotransduction and largely due to the fact that progenitors were preferentially transduced and then disappeared during the first weeks of culture. A representative flow cytometry analysis of EGFP expression from mPB cells is shown in Figure 3Go.



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  		Figure 2. Maintenance of EGFP expression during long-term culture. EGFP was first measured 72 hours after transduction (day 6). Nonadherent cells from weekly semidepopulations of long-term cultures of TEE-transduced CD34+ mPB cells were then analyzed by flow cytometry (FL-1 channel). Control untransduced mPB cells showed less than 1% fluorescent cells. MOI values are final MOI. Values are means from two to three separate experiments. For CB cells, MOI were: 20; {blacksquare} 50; 100; {square} 400; for mPB cells, MOI were: {bullet} 50; 100; 200; {circ} 400.

 


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  		Figure 3. Flow cytometric analysis of transduced cells. Representative flow cytometric analysis of EGFP in CD34+ or CD34+/38-/lin- -derived cells after lentiviral gene transfer. Cells were transduced at an MOI of 100, with or without prestimulation with IL-3, SCF, FL, and TPO. Ninety-six hours after the second transduction, cells were harvested and stained with antihuman CD34-PC-5 antibody. Control cells were stained with an isotype control. EGFP fluorescence was detected in channel FL-1. Coordinates were set to locate >99% ofisotype events in the lower left quadrant.

 
Effect of Prestiniulation on Transduction Efficiency in CD34+ mPB Cells
In a second series of experiments, prestimulation was carried out over a prolonged period of 18 hours, with the same RM+CK medium. Results presented in Figure 4AGo (gray bars) show high gene transfer efficiencies in the total and CD34+ cell populations with 85.9% ± 6.9% and 85.5% ± 7.2% of EGFP+ cells, respectively. Levels of gene transfer in CFC-derived colonies doubled when cells were cultured for 18 hours with cytokines prior to transduction, reaching 81.7% ± 10.5% of EGFP colonies when only 41.3% ± 3.5% were transduced without stimulation (Fig. 1CGo and open bars in Fig. 4AGo ). Finally, when looking at LTC-IC-derived colonies, we found a 4.2-fold increase in gene transfer efficiency (62.8% ± 5.4% EGFP+ colonies with prestimulation versus 15% without prestimulation). In liquid culture, the percentage of transduced cells decreased to finally stabilize around 54% transduction after 1 month of culture, which still represented a 2.2-fold increase compared with nonprestimulated CD34+ cells (with only 24% of cells expressing EGFP) (Fig. 4BGo). Southern blot analysis was performed with DNA purified from prestimulated mPB CD34+ transduced cells after 14 days of culture. The expected size of the proviral band (1.8 kb) was found. The percentage of CD34+ cells in this sample was 78%. The mean copy number per cell was estimated at 3.0 for total cells and 3.8 for transduced cells (Fig. 5Go).



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  		Figure 4. Transduction efficiency after lentiviral gene transfer in mPB CD34+ and CD34+/38-/lin- cells after cytokine prestimulation. All cells were transduced twice at an MOI of 50 (final MOI of100) and analyzed 72 hours after the last transduction. Results for nonprestimulated mPB CD34+ cells (open bars for part A and {circ} for part B) are those presented in Figure 1Go. Results for 18-hour-prestimulated CD34+ cells (gray bars and {square}) are from three separate experiments performed on the same mPB. Results for CD34+/38-/lin- cells (black bars and {blacktriangleup}) are from three individual experiments performed on three distinct mPBs. A) Percentage of EGFP expression in the bulk population, CD34+ expressing cells, CFCs, and LTC-ICs. Values are means ± standard deviations from three independent experiments. B) Follow-up of gene transfer in long-term cultures. Values are means ± standard deviations from three to four separate experiments. Enclosed is the relative fluorescence intensity (RFI) in transduced CD34+/38-/lin- mPB cells measured at the same time points as the percentage of transduced cells. Values are the fold difference in mean fluorescence intensity of events above the threshold (determined with untransduced cells) compared with events below the threshold (background fluorescence). *Significant difference versus unstimulated CD34+ cells (p < 0.05). **Significant difference (p < 0.05) versus 18-hour-stimulated CD34+ cells. The Mann-Whitney test was used.

 


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  		Figure 5. Proviral integration analysis. Genomic DNA from untransduced and TEEW-transduced CD34+ mPB cells was digested with EcoRI and KpnI, blotted, and hybridized with the WPRE probe as shown in Figure 1Go. Lanes A–D: DNA from untransduced cells mixed with decreasing amounts of TEEW plasmid (166.7 to 16.67 pg, i.e., 10 to 1 copy, respectively). Lane E: DNA from untransduced cells. Lane F: molecular weight markers. Lane G: DNA from TEEW-transduced cells (78% GFP+ cells). The 1.8-kb band corresponds to the integrated provirus.

 
Lentiviral Gene Transfer Efficiency in mPB CD34+/38-/lin- Cells
With this optimized protocol, we finally investigated gene transfer in CD34+/38-/lin- cells isolated from mPB. This fraction accounted for 0.1%–1% of the original CD34+ fraction. The lentiviral vector used in these experiments was TEEW, in which the WPRE sequence was inserted downstream of EGFP in the TEE vector, in order to sustain long-term high-level transgene expression. From three different mPB samples, we obtained very high gene transfer levels in the bulk population, with 90.3% ± 2.4% of EGFP+ cells, and similar gene transfer rates in cells that still expressed CD34 after 6 days of culture (with 86.9% ± 5.0% of these cells expressing the transgene) and also in CFC-derived colonies (transduction efficiency of 86.6% ± 3.2%) (Figs. 3Go and 4AGo ; black bars). When looking at LTC-IC-derived colonies, gene transfer efficiency remained at a very high level with 87.7% ± 9.4% of colonies expressing EGFP. EGFP expression was also analyzed in long-term cultures for up to 10 weeks after transduction by flow cytometric analysis of cells recovered from weekly semidepopulations. Results show a remarkable stability of both the percentage of transduced cells and the level of expression, despite a slight decrease in the first 3 weeks (Fig. 4BGo ).


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The principal aim of gene therapy using HSCs as target cells is to provide long-term genetic correction of a sufficient number of life-long repopulating cells. This includes genomic integration and sustained expression of the transgene, maintenance of life span, self-renewal capacities, and multilineage differentiation potential of the transduced cells and/or their progeny. Despite nearly two decades of efforts, the expected therapeutic benefits mainly remained unfulfilled.

Moloney murine leukemia virus-derived retroviral vectors have been widely used in gene transfer experiments. But, despite many improvements in the vectors themselves and in the transduction procedures, a major block persists because these retroviruses are not efficient at transducing nondividing cells [11]. Unfortunately, HSCs are mostly quiescent cells, and attempts to stimulate these cells to enter the cell cycle have resulted in higher percentages of transgene-expressing cells but also in decreased long-term repopulating capacities. Moreover, a majority of studies investigated gene transfer in CB, but few investigators have worked with mPB. Reported results show a discrepancy in transduction efficiency in mPB cells compared with cells from CB [14]. The reason for these differences is not yet known, but several reports suggest that stem cells derived from mPB may be in a deeper state of dormancy than cells from CB or BM [21 ]. Thus, prolonged stimulation of these cells might be required to allow retroviral integration in mPB HSCs with, in counterpart, a detrimental effect on the maintenance of the long-term reconstituting ability of these cells [22, 23]. Despite these limitations, and in the particular context of a selective advantage of the transduced cells, gene therapy with retroviral vectors was achieved recently in X-linked severe combined immunodeficiency (SCID) by {gamma}c gene transfer into CD34+ cells [24, 25]. Similar results have also been obtained with other forms of SCID [26–28]. However, for most of the monogenic diseases, there is no selective advantage of corrected cells, and the main problem resides in the very low percentage of corrected cells (usually <1%) in blood after several months. This is the case for adrenoleukodystrophy [29], Gaucher disease [30], and chronic granulomatous disease [31].

An alternative has recently emerged with the use of lentiviral vectors derived from HIV-1 or other types of lentiviruses such as simian immunodeficiency virus or feline immunodeficiency viruses. Modified lenti vectors with internal promoters permitted very efficient and stable transgene expression in HSCs [32–34] without adverse effects due to downregulation of long terminal repeat promoters that often occur with retroviral vectors. The latest third-generation vectors now provide very efficient transduction levels in many types of cells and stable transgene expression. But differences in transduction efficiency between CB and mPB still persist, particularly in the most primitive cells as demonstrated by lower overall results from studies comparing mPB cells with CB cells [35–37]. Achieving long-term pancellular expression implies the use of high liter vectors, leading to multiple proviral integrations [38, 39]. However, this situation increases the risk of insertional mutagenesis, promoting the appearance of a malignant phenotype of the cell. Unfortunately, this was demonstrated recently in the mouse [40] and human [41], leading to a new reflection on how to better define this risk [42].

In this report, we first compared the effects of increasing MOI on transduction efficiency in both sources of cells. CB and mPB CD34+ cells were transduced with MOI ranging from 20–400 using a VSV-G pseudotyped SIN-lentiviral vector containing the EGFP reporter gene driven by the human elongation factor 1{alpha} promoter. Transductions were carried out using a simple protocol with two transductions in the presence of the fibronectin fragment CH-296, in serum-free medium supplemented with IL-3, FL, SCF, and TPO. Results were different between CB and mPB, with CB cells being efficiently transduced even with low MOI, while mPB cells in the same conditions demonstrated lower gene transfer rates, especially with an MOI under 100. A second interesting feature is the fact that CB cells were not significantly better transduced at an MOI of 400 than at an MOI of 20, whereas the percentage of transduced mPB cells increased between these two conditions. Finally, it appears that increasing MOI is not sufficient, in this experiment, to overcome a threshold in gene transfer efficiency for mPB cells. We have reached a plateau for this source of cells, with 25% of cells being refractory to lentiviral infection under the present conditions. Results of transduction in CD34+ cells, CFCs, and LTC-ICs showed the same differences between the two sources of cells.

Even if lentiviral vectors have been shown to transduce nondividing cells, recent reports have showed that, although CD34+ cells in G0 can be transduced, CD34+ cells in G1or S/G2/M are much more efficiently transduced [21]. We then chose to prestimulate mPB CD34+ cells for 18 hours before transduction in the presence of SCF, FL, TPO, and IL-3. The transduction procedure consisted of two infections at 24-hour intervals (MOI of 50 each time) without the fibronectin fragment CH-296. We demonstrated a significantly greater gene transfer efficiency compared with unstimulated (i.e., <3 hours of culture in serum-free medium with cytokines) cells, particularly in CFCs and LTC-ICs with up to 63% of colonies derived from LTC-ICs expressing EGFP. These transduction rates were two- to fourfold higher than with unstimulated mPB cells, for CFCs and LTC-ICs, respectively. These high gene transfer rates were obtained with a reasonable number of integration sites (a figure in the region of 3–4 copies per cell), thus limiting the risk of insertional oncogenesis.

The vast majority of CD34+ cells do not have multilin-eage potential. We next wanted to investigate gene transfer in a stem/progenitor-cell-enriched population, the CD34+/ 38-/lin- cell subset [20, 43–45]. There are two advantages of using such an enriched population: first, the number of cells to be genetically modified is divided by a factor of 10 to 100, facilitating the manipulations of cells; second, the risk of insertional oncogenesis is limited by the low number of cells that integrate the provirus. Using the cytokine prestimulation protocol we had previously established for mPB CD34+ cells, we analyzed gene transfer efficiencies in total and CD34+ cells, CFCs, and LTC-ICs, in long-term liquid cultures for up to 10 weeks. We observed very high transduction efficiencies, with >85% of EGFP+ cells (bulk population and CD34+ cells) or colonies derived from CFCs and, more importantly, from LTC-ICs. These results demonstrate a nearly total transduction of a subset of very primitive cells, with prestimulation limited to 18 hours and only two infections at an MOI of 50. These results, together with other convergent reports [35–37], demonstrate the feasibility of very efficient gene transfer in hematopoietic/stem progenitor cells isolated from adult sources using lentiviral vectors. However, the behavior of these transduced cells in vivo should be addressed to determine the impact of this type of gene transfer protocol on the fate of the most primitive cells capable of reconstituting multilineage hematopoiesis in irradiated recipients. This improvement in the efficiency of gene transfer with lentiviruses could contribute to the development of successful clinical protocols for diseases of the hematolymphoid system.


   ACKNOWLEDGMENT
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We thank P. Charneau for donating the TRIP{Delta}AU3-EF1{alpha} vector, J. Reiffers, and B. Dazey for the CD34+ mPB cells, and Catherine Verret for statistical analysis. This work was supported by grants from Association Française centre les Myopathies (AFM) and Conseil Régional d’Aquitaine.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received December 13, 2002; accepted for publication May 6, 2003.



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