Reconstitution of the myeloid and lymphoid compartments after the transplantation of autologous and genetically modified CD34+ bone marrow cells, following gamma irradiation in cynomolgus macaques

Background Prolonged, altered hematopoietic reconstitution is commonly observed in patients undergoing myeloablative conditioning and bone marrow and/or mobilized peripheral blood-derived stem cell transplantation. We studied the reconstitution of myeloid and lymphoid compartments after the transplantation of autologous CD34+ bone marrow cells following gamma irradiation in cynomolgus macaques. Results The bone marrow cells were first transduced ex vivo with a lentiviral vector encoding eGFP, with a mean efficiency of 72% ± 4%. The vector used was derived from the simian immunodeficiency lentivirus SIVmac251, VSV-g pseudotyped and encoded eGFP under the control of the phosphoglycerate kinase promoter. After myeloid differentiation, GFP was detected in colony-forming cells (37% ± 10%). A previous study showed that transduction rates did not differ significantly between colony-forming cells and immature cells capable of initiating long-term cultures, indicating that progenitor cells and highly immature hematopoietic cells were transduced with similar efficiency. Blood cells producingeGFP were detected as early as three days after transplantation, and eGFP-producing granulocyte and mononuclear cells persisted for more than one year in the periphery. Conclusion The transplantation of CD34+ bone marrow cells had beneficial effects for the ex vivo proliferation and differentiation of hematopoietic progenitors, favoring reconstitution of the T- and B-lymphocyte, thrombocyte and red blood cell compartments.


Background
Gene therapy strategies hold promise for the treatment of hematopoietic disorders. All hematopoietic lineages, including polymorphonuclear cells, monocytes, lymphocytes and natural killer cells, and hematopoietic stem cells (HSC) -which are capable of self-renewal and pluripotent differentiation -have been targeted for transduction with therapeutic genes. Most diseases for which gene therapy could be proposed require stable and longlasting transgene expression for efficacy. Retroviral vectors present the major advantage of integrating the transferred DNA stably into the genome of target cells, which is then passed on to progeny. However, they cannot infect and integrate into non dividing cells [1]. Most HSC are quiescent [2], respond slowly to stimulation [3][4][5][6][7] and tend to differentiate and lose their repopulating capacity upon stimulation [3,[8][9][10][11]. Lentiviral vectors can be used to transduce cells in growth arrest [12]in vivo and ex vivo [13], thanks to interaction of the preintegration complexcomposed of viral VPX and integrase proteins -with the nuclear pore complex [14]. Vectors derived from HIV-1 [15,16], HIV-2 [17], FIV [18] and equine infectious anemia virus (EIAV)have been tested [19].
Methods for transferring genes into hematopoietic cells must be tested in relevant animal models before their application to humans [20,21]. Studies in nonhuman primates (NH)P provide an ideal compromise, because these species are phylogenetically closely related to humans and a high level of nucleotide sequence identity is observed between the genes encoding many hematopoietic growth factors and cytokines in these mammals and their counterparts in humans [22]. Moreover, hematopoiesis in macaques is very similar to that in humans, and the HSC biology of macaques is much more similar to that of humans than is that of rodents, making macaques good candidates for hematopoietic stem cell engraftment studies [23][24][25][26]. In addition, testing lentiviral based gene transfer strategies need to be assessed in species that are susceptible to lentivirus induced disease. Or particular interest are the Feline immunodeficiency virus (FIV) infection which causes a clinical disease in cats that is remarkably similar to HIV disease in human [27][28][29][30] and experimental infection of macaques with the simian immunodeficiency virus (SIV) reproducing both chronic infection and an AIDS-like disease very similar to those observed in human patients infected with HIV. Despite the theoretical advantages of lentiviral vectors over oncoretroviral vectors, non human primate lentiviruses clearly have pathogenic properties [31]. The use of lentiviral vectors derived from potentially pathogenic primate lentiviruses, such as SIV, therefore continues to raise serious clinical acceptance concerns. SIV-based vectors, such as SIVmac239 [31,32] and SIVmac251 [33,34], may pro-vide a unique opportunity to test the safety and efficacy of primate lentiviral vectors in vivo.
Recent improvements in the efficiency of gene transfer to NHP repopulating cells [11,35,36] have provided new opportunities to follow the progeny of each primitive progenitor and stem cells directly in vivo, using retroviral marking to track individual progenitor or stem cell clones [37]. Clinically relevant levels (around 10%) of genetically modified cells in the peripheral blood have been achieved by ex vivo gene transfer into HSC and the autologous transplantation of these cells into macaques [37]. Successful and persistent engraftment (up to six months) has also been reported in non human primates with primitive CD34 + progenitors genetically modified with a murine retrovirus vector encoding the murine CD24 gene as a reporter gene [38]. In both trials, marked cells of multiple hematopoietic lineages were identified in the blood: granulocytes, monocytes and B and T cells, including naive T lymphocytes [37,38]. The efficacy of HSC gene transfer could theoretically be improved by the use of newly developed retroviral or lentiviral vectors. Particles bearing an alternative envelope protein, such as that of the feline endogenous virus (RD114), have been shown to be superior to amphotropic vectors for the transduction of NHP stem cells followed by autologous transplantation [39,40].
We report here the results obtained in vitro and in vivo in an experiment assessing the efficacy and safety of a gene transfer protocol based on the transduction of simian CD34 + bone marrow cells with a minimal SIVmac251derived lentiviral vector. This system is based on the VSVgpseudotyped SIV vector encoding enhanced green fluorescent protein (eGFP) under control of the phosphoglycerate kinase (PGK) promoter. Most immature CD34 + hematopoietic cells capable of initiating long-term culture (LTC-IC) were efficiently transduced, and eGFP-positive cells were detectable in vivo in all animals more than one year after transplantation.

Animals
Male cynomolgus macaques (Macaca fascicularis), weighing between 3 and 6 kg were imported from Mauritius and housed in single cages within level 3 biosafety facilities, according to national institutional guidelines (Commission de génie génétique, Paris, France). All experimental procedures were carried out in accordance with European guidelines for primate experiments (Journal Officiel des Communautés Européennes, L358, December 18 1986).

Immunoselection of non human primate CD34 + bone marrow progenitor cells
Bone marrow mononuclear cells were obtained from the iliac crest or by aspiration from the humerus and isolated by standard Ficoll density-gradient centrifugation (MSL2000, Eurobio, Les Ulis, France). Cells were washed twice in phosphate-buffered saline (PBS, Eurobio, Les Ulis, France) and resuspended in 1% FCS (Fetal Calf Serum; Bio West, France) in PBS. The cellular fraction was then enriched in CD34 + cells by positive immunomagnetic selection, using beads coupled to a specific antibody (clone 561; Dynabeads M-450 CD34, Progenitor Cell Selection System, Dynal, Oslo, Norway), according to the manufacturer' s instructions. Immunoselected CD34 + cells were stained with a specific PE-conjugated anti-CD34 antibody (clone 563; Pharmingen, Becton Dickinson, California, USA) and analyzed by flow cytometry (LSR, Becton Dickinson, California, USA) to evaluate the level of enrichment. All preparations contained more than95% CD34 + cells, with a mean value of 97% ± 1% (n = 12) for in vitro assays and 96% ± 1% (n = 4) for in vivo assay.

Lentiviral vector
Two SIV-derived vectors were produced, one for in vitro studies and the other for in vivo studies: 1) pRMES8 is a minimal packaging-competent SIVmac251-based vector [34]. It contains the enhanced green fluorescent protein (eGFP) marker gene under control of the mouse phosphoglycerate kinase (PGK) promoter, placed between the SIVmac251 LTRs and leader sequences. It carries the SIVmac251 RRE region and minimal sequences of the gag and pol genes encompassing central polypurine tract/central termination sequence (cPPT/CTS) regions (figure 1A). pRMES8 was used for in vitro assays investigating the susceptibility of CD34 + cells from primate bone marrow to transduction with SIVmac251-derived vectors. 2) For in vivo assays, we used pGASE; this plasmid is an optimised version of pRMES8, with a 3'-SIN-LTR for safety and insertion of an exon splicing enhancer (ESE) upstream the PGK promoter to increase titer [41] pSIV3 + is the packaging plasmid derived from the BK28 molecular clone of SIVmac251, as described elsewhere [33]. Briefly, the pSIV3 + gag/pol expression plasmid Schematic representation of SIV-derived SIN vector, helper construct and VSV-g encoding plasmid Figure 1 Schematic representation of SIV-derived SIN vector, helper construct and VSV-g encoding plasmid. An SIVmac251-derived vector was produced by cotransfecting 293T cells with three plasmids: A. a plasmid pGASE containing the eGFP gene under control of the PGK promoter; B. a plasmid pSIV3+ containing viral genes; C. a plasmid pGREV containing the VSV envelope gene. Cis genetic elements are symbolized with white boxes, whereas promoters and genes are depicted by shadowed boxes. pCMV, early cytomegalovirus promoter; pPGK, mouse phosphoglycerate kinase-1 promoter; RRE, REV-responsible element; SA, SIV Rev/Tat splice acceptor; cPPT and PPT, central and 3' polypurine tracks, respectively; GFP, the gene encoding the enhanced green fluorescent protein; LTRsin, partially U3 deleted 3'LTR; LG, leader and a 5' GAG region. pGREV was used for pseudotyping. It is a bicistronic expression construct encoding the vesicular stomatitis virus glycoprotein (VSV-g) and the REV regulatory protein, linked by an EMCV IRES. Expression of this cassette, which contains the rabbit β-globin intron II and polyadenylation (pA) sequences (figure 1C), is driven by the constitutive CMV promoter.

Production of SIV vectors
293T cells were plated at a density of 4.0 × 10 5 cells per well (in 6-well plates) on the day before transfection. Cells were transfected as previously described [42]. SIV vectors were produced by cotransfection with three plasmids: the SIV plasmid vector (pRMES8 or pGASE)(1.7 μg), the helper plasmid, pSIV3 + , encoding Gag-Pol and regulatory proteins other than Env and Nef (1.7 μg) and the envelope glycoprotein-encoding plasmid pGREV (2.2 μg). The transfection medium was replaced after 16 hours of incubation. Virus-containing medium was collected 40 hours after transfection, clarified by centrifugation for 5 minutes at 800 g, and passed through a filter with 0.45 μm pores. For high-titer preparations, SIV vectors were concentrated by ultracentrifugation at 110,000 g for 2 hours. The viral pellet was resuspended by incubation for 2 hours at 4°C in phosphate-buffered saline supplemented with 1% glycerol [34].
For determination of the infectious titer, sMAGI cells were seeded at a density of 4 × 10 5 cells/ml in six-well plates one day before transduction in DMEM medium (Life Technologies Inc., Berlin, Germany) supplemented with 10% fetal bovine serum (FBS) (Gibco BRL, Grand Island, New York, USA), polybrene (6 μg/ml) (Sigma, Saint Louis, USA) and an antibiotic mixture (5 mg/ml penicillin; 5 mg/ml streptomycin; 10 mg/ml neomycin; Gibco BRL, Grand Island, New York, USA). The cells were cultured for one day, and we then added serial dilutions of virus preparations and incubated the plates for a further four hours. Cells were then washed in DMEM (Life Technologies Inc., Berlin, Germany). Transduction rates was

Myeloid differentiation of CD34 + cells
Following the coculture of CD34 + cells with lentiviral vector, part of the cell culture was fixed in CellFix solution (Becton Dickinson, Erembodegem, Belgium) for evaluation of the rate of transduction of undifferentiated CD34 + cells. Part of the cell culture was cultured for 14 days in 35 mm Petri dishes containing semi-solid medium (Methocult GF H4434, Stem Cell Technologies, Meylan, France) composed of Iscove's MDM medium supplemented with 1% methylcellulose, 30% fetal bovine serum, 10 -4 M 2mercaptoethanol, 2 mM L-glutamine, 50 ng/ml rhSCF, 10 ng/ml rhGM-CSF, 10 ng/ml rhIL-3 and 3 IU/ml rhEPO. Cells were cultured at a density of 10 4 cells/ml (in triplicate) at 37°C, under an atmosphere containing 5% CO 2 , to allow the myeloid differentiation of colony-forming cells (CFC).
The remaining cells were cocultured in 96-well plates for 35 days at 37°C, under an atmosphere containing 5% CO 2 , on a layer of stromal cells of the murine fibroblastic cell line M2-10B4, in a medium composed of αMEM supplemented with 12.5% horse serum (HS), 12.5% FBS, 2 mM L-glutamine, 10 -4 M 2-mercaptoethanol, 0.16 M Iinositol and 16 μM folic acid (Myelocult H5100, Stem Cell Technologies, Meylan, France) and 10 -6 M hydrocortisone. Cells were cultured at a concentration of 10 3 cells per well (24 wells per condition per monkey), to allow long-term culture-initiating cells (LTC-IC) to undergo myeloid differentiation to generate progenitor cells or CFC. The CFC were cultured for 14 days on semi-solid medium, as described above, to allow their myeloid differentiation into more mature cells.

AZT pretreatment of immunoselected CD34 + cells
CD34 + cells were treated with AZT before transduction, to inhibit transduction due to reverse transcription of the lentiviral vector genome. Immunoselected CD34 + cells were cultured overnight in the proliferation medium described above, with AZT concentrations of 0, 10 -7 , 10 -6 and 10 -5 molar. The cells were washed twice and transduced with the lentiviral vector, according to the protocol described above. The real percentage of GFP-positive cells resulting from reverse transcription of the lentiviral vector was thus determined by subtracting the percentage of GFP-positive cells obtained after treatment with a saturating dose of AZT, from the percentage of GFP-positive cells obtained in the absence of AZT treatment.

Fluorescence microscopy
After transduction and myeloid differentiation in semisolid medium, the colonies formed by AZT-treated CFC were observed by fluorescence microscopy (Axiovert S100, Zeiss) using a magnification factor of 100. Fluorescence microscopy was used to detect GFP in each colony subtype, making it possible to determine the percentage of the colonies positive for GFP. We considered all colonies containing GFP-producing cells to be GFP-positive. Images were analyzed with Adobe Premiere and Adobe Photoshop software (Adobe Systems Inc., San Jose, CA, USA).

Gamma irradiation
Eight animals were sedated with ketamine (Imalgène; 10 mg/kg, i.m.), Rhône-Mérieux, France) and placed in a restraint chair. They received myeloablative conditioning, in the form of total body exposure to 60 Co gamma rays with an anterior unilateral direction. A total midline tissue dose of 6 Gy was delivered at a rate of 25.92 cGy/minute. Dosimetry was performed, with 100 μL ionization chambers placed in paraffin wax cylindrical phantoms of a similar size and orientation to the seated animal.

Transplantation of modified CD34 + bone marrow cells
After the coculture of CD34 + cells with the lentiviral vector, four animals underwent intramedullary infusion, of whole immunoselected CD34 + cells into both humeri (Table 1).

Clinical support
All animals received clinical support in the form of antibiotics and fresh irradiated whole blood, as required. An prophylactic antibiotic regimen was initiated when leukocyte count fell below 1,000/μl and continued daily until it exceeded 1,000/μl for three consecutive days: 1 ml/10 kg/ day Bi-Gental ® (Schering-Plough Santé Animale) and 1 ml/10 kg Terramycin ® (Pfizer). Fresh, irradiated (25 Gy; 137 Cs gamma radiation) whole blood (approximately 50 ml/transfusion) from a random donor pool was administered if platelet count fell below 20,000/μl and hemoglobin concentration was less than 6 g/dl.

Efficient transduction of cynomolgus macaque CD34 + bone marrow cells
We first assessed, in vitro, the efficiency with which a SIVmac251-derived vector transduced CD34 + hematopoietic cells from macaque bone marrow (BM). We harvested BM cells from the iliac crests of 12 different animals. CD34 + cell preparations with a purity of 97% ± 1% were obtained by immunomagnetic purification. The CD34 + cells were then transduced by coculture for 24 h with the lentiviral vector (MOI = 100) in medium supplemented with SCF, Flt3-L, IL-3 and IL-6. The vector used (pRMES8) was derived from SIVmac251 and contains the eGFP reporter gene under control of the phosphoglycerate kinase (pGK) promoter ( Figure 1). Transduction efficiency (Figure 2A and 2B), as evaluated by flow cytometry analysis of eGFP expression at 24 h, was 41% ± 9% on average (n = 12). After 24 hours of culture with the lentiviral vector, some of the purified CD34 + cells were cultured for 14 days in semi-solid medium containing SCF, GM-CSF, IL-3 and EPO to allow the myeloid differentiation of colony-forming cells (CFC), whereas some cells were cocultured for 35 days on a layer of murine fibroblasts of the M2-10B4 cell line and were then cultured for 14 days on semi-solid medium containing SCF, GM-CSF, IL-3 and EPO, for the identification of long-term cultureinitiating cells (LTC-IC). Transduction had no effect on the clonogenic capacity of CD34 + cells: the mean number of colonies was 41 ± 10 for non transduced cells and 44 ± 12 for pRMES8-transduced cells (12 animals tested, P = 0.60 (Mann & Whitney test)). Similar results were obtained for LTC-IC, with 19 ± 3 colonies obtained for non transduced cells and 19 ± 3 for transduced cells (n = 12; P = 0.79 (Mann & Whitney test)). Transduction rates did not differ significantly between CFC and LTC-IC (P = 0.4884 (Wilcoxon test), n = 12), with 18% ± 7% and 19% ± 7% of colonies, respectively, eGFP-positive. However, in both cases, the percentage of eGFP-positive cells was significantly lower than that observed 24 hours after transduction (P < 0.0001 (Wilcoxon test)). This apparent discrepancy between analyses carried out at 24 h and analyses on CFC or LTC-IC may be due to the eGFP protein present in viral particles and incorporated into the cell cytoplasm during the coculture period. The proportion of cells producing eGFP shortly after transduction was reduced by 25% ± 15% ( Figure 2C) if 10 -6 M AZT was added to cocultures of CD34 + BM cells and lentiviral vector (MOI = 100). Untreated CFC cultures gave percentages of eGFP-producing cells similar to those observed before differentiation (26% ± 5%) ( Figure 2D). No fluorescence was detected after myeloid differentiation of the AZTtreated CFC (n = 3), confirming that eGFP detection resulted from the production of this protein from integrated vector.
Mosaicism was observed in eGFP gene expression in several colonies (Figure 3). Indeed, eGFP was detected in 56% ± 4% of colonies, whereas only 26% ± 5% of individual cells were eGFP-positive. These results suggest that, on average, only 47% of cells from a single colony contained the SIV vector.

Transplantation of autologous BM CD34 + cells transduced by SIV-based vector into cynomolgus macaques
We explored the capacity of autologous CD34 + BM cells transduced ex vivo with a lentiviral vector to engraft efficiently into macaques after total body irradiation (TBI) with a gamma source at the sublethal dose of 6 Gy. Three groups of 4 animals were used: 1) In Group 1, macaque CD34 + BM cells (96% ± 1% pure on average) were obtained from the two humeri before gamma irradiation (Table 1). These cells were cocultured, as described above, with pGASE, which is an improved version of pRMES8. Indeed, a mean transduction efficiency of 72% ± 4% was obtained (n = 4) at 24 hours and 37% ± 10% of CFC produced eGFP. Two days after gamma irradiation, 1.4 × 10 6 to 2.9 × 10 6 CD34 + cells per kg were injected into both humeri of macaques (Table 1); 2) Group 2 included irradiated (6 Gy) macaques that did not undergo cell transplantation: 3) Group 3 included 4 non irradiated animals, which were used as controls, with a similar bleeding frequency.

Reconstitution of hematopoietic cells in vivo
Following total-body irradiation with 6 Gy, transfusion and an antibiotic regimen were required to ensure that all the animals survived. However, one animal from group 1 (7036) died on day 40 due to profound pancytopenia ( Figure 4). This macaque received the smallest number of autologous and transduced CD34 + BM cells. All other ani-mals from groups 1 and 2 were studied from days -1 to 471 after gamma irradiation. Controls were followed over the same period.
Radiation rapidly induced severe anemia in all animals (data not shown). A significant decrease in the number of polymorphonuclear cells in the periphery was observed, starting on day 1 after irradiation ( Figure 4). No significant difference was observed between the animals of groups 1 and 2 in terms of the minimum number of cells (821 ± 226 cells/μl for group 1 and 658 ± 107 cells/μl for group 2, P = 0.3768 (Mann & Whitney test)) or the time at which that minimum occurred (6 ± 5 days for group 1 and 7 for group 2, P = 0.4795 (Mann & Whitney test)). Lymphocyte counts also decreased in all macaques by day 1 after gamma irradiation (Figure 4), falling to a minimum of 220 ± 107 lymphocytes/μl on day 18 ± 12 in group 2 and of 347 ± 62/μl on day 11 ± 12 in transplanted animals (group 1). Animals undergoing transplantation tended to display less severe lymphopenia, but no statistical difference was observed between the two groups of irradiated animals in terms of the day on which minimum   Whitney test)). This thrombocytopenia required one transfusion in all animals (other than animal 7036, which needed two transfusions) of both groups. However, platelet reconstitution seemed to be correlated with the dose of CD34 + cells infused, the speed of reconstitution increasing with the number of CD34 + cells injected (macaque 6653).

Reconstitution of bone marrow clonogenic activity
We determined the effects of CD34 + bone marrow cell transplantation following gamma irradiation on the ex vivo proliferation and differentiation of hematopoietic progenitors. Before gamma irradiation, a mean of 40 ± 9 and 38 ± 6 colonies was observed for groups 1 and 2, respectively ( Figure 5). Colony number decreased significantly (P < 0.0001 (Wilcoxon test)) by day 7 in all animals. In both groups, clonogenic activity was detected by day 43 after gamma irradiation with reconstitution significantly better in the animals undergoing transplantation than in those that did not undergo transplantation (P = 0.0009 (Mann & Whitney test)).

Presence of eGFP-positive cells in bone marrow and peripheral blood
Cells with integrated SIV-vector DNA were detected by PCR (Table 2)  Effect of irradiation and transplantation on polymorphonuclear cell, lymphocyte and thrombocyte counts Figure 4 Effect of irradiation and transplantation on polymorphonuclear cell, lymphocyte and thrombocyte counts. All animals were followed during the weeks preceding the study, and for more than 240 days after the irradiation. We carried out hematological analysis including blood cell counts with an automated hemocytometer (Coulter Corporation, Miami, USA).    Flow cytometry analysis demonstrated the presence of eGFP-producing cells among peripheral blood mononuclear cells in myeloid and lymphoid lineges of monkey 6896 ( Figure 6). Peripheral blood cells were sorted on the basis of eGFP production, with the aim of characterizing the phenotype of populations of cells expressing the transgene in more detail. We found that 61% of eGFP-positive cells were CD11b-positive,5% of these cells appeared to be CD14+ monocytes, 14% were CD20 + B cells and 10% were CD3+ T cells, 23% of which expressed CD8 and 77% expressed CD4 (data not shown).

Discussion
The aim of this work was to study reconstitution of the myeloid and lymphoid compartments after the autologous transplantation of genetically modified CD34 + bone marrow cells into cynomolgus macaques previously subjected to gamma irradiation.
We first assessed, in vitro, the efficiency with which a SIVmac251-derived vector transduced macaque CD34 + hematopoietic bone marrow cells. These vectors are similar to those derived from HIV. However, SIV-derived vectors clearly outperform HIV-derived vectors in simian cells. In fact, HIV-1 fails to replicate in simian cells because of an early postentry block [43,44], and Kootstra et., al showed that the viral determinant involved in postentry restriction of HIV-1 replication in simian cells is located at or near the cyclophilin A (CyPA) binding region of the capside protein [45]. The hydrophobic pocket of cyclophilin A (CypA) makes direct contact with an exposed, proline-rich loop on HIV-1 capsid (CA) and renders reverse transcription complexes resistant to an antiviral activity in human cells. A CypA fusion with TRIM5 (a member of the tripartite motif family) that is unique to New World owl monkeys also targets HIV-1 CA, but this interaction potently inhibits infection. A similar block to HIV-1 infection in Old World monkeys is attributable to the α isoform of the TRIM5 orthologue in these Recovery of bone marrow clonogenic activity species and using RNA interference techniques, Berthoux et., al demonstrated that CypA inhibits HIV-1 replication in these cells because it is required for CA recognition by TRIM5α [46]. SIV vectors can also efficiently transduce human cells [33,47], and may therefore prove a useful alternative to HIV-1-based vectors, at least in the early phase of preclinical testing of lentivirus vectors. We found that the proportion of eGFP-positive cells obtained before myeloid differentiation (mean value of 30%) was similar to that obtained with CD34 + cells from human donors transduced with lentiviral [48][49][50][51], retroviral [52][53][54], AAV [55], or adenovirus/AAV-derived [56] vectors. However, it is possible to increase the transduction rate, such that 90% transduced human CD34 + cells are obtained from cord blood, 80% from bone marrow and 75% from G-CSF mobilized peripheral blood [57]. We analyzed transduction in two types of assay, based on committed (CFC) and primitive (LTC-IC) hematopoietic progenitors, as analyses of the transduction of committed progenitors only bear little relation to the transduction efficiency for stem cells and less differentiated cells in the long term.
After myeloid differentiation, eGFP + cells were detected, in similar proportions, in CFC on day 15 and LTC-IC on day 50 after transduction, indicating that the vector was able to transduce progenitor cells and most immature hematopoietic cells with a similar efficiency. Similar results have been reported for stimulated human CFC and LTC-IC, which were found to be transduced with similar efficiency by a lentiviral vector based on HIV-1 [58]. In this previous study, significant resistance to lentiviral transduction was reported in unstimulated primitive human cells. These results may explain why, in our study, the use of cytokines during transduction made possible the genetic modification of LTC-IC, which are quiescent. Cytokine treatment may have led to these cells entering the cell cycle, facilitating transduction. This result confirms the greater efficiency of lentiviral vectors than of retroviral vectors for the transduction of CD34 + cells. Nevertheless, in our study, only half as many eGFP-positive cells were obtained after differentiation as were obtained from undifferentiated CD34 + cells. Similar observations have been made with MLV-transduced progenitor cells from human Flow cytometry analysis of hematopoiesis reconstitution Figure 6 Flow cytometry analysis of hematopoiesis reconstitution. Animal transplanted with autologous CD34 + bone marrow cells transduced with an SIV-based vector. eGFP-positive cells present in P1 and P2 were analyzed by immuno-staining to identify the subpopulations of eGFP-positive cells in peripheral blood. CD20-PerCP-Cy5, CD14-PE, CD11b-APC and CD3-APC staining were used to identify the B-lymphocyte, monocyte, granulocyte and T-lymphocyte subpopulations. donors [59]. We demonstrate here that these differences may be accounted for by the pseudotransduction detected at 24 h of incubation with the vector, confirming the results reported with CD34 + cells in studies using VSVgpseudotyped MLV-derived [60] or lentivirus-derived vectors [51]. It has been suggested that pseudotransduction may result from VSVg-pseudotyping due to membrane fusion efficiency being higher than the rate of integration of the transgene [61]. Nevertheless, most lentiviral vectors have been generated with VSV-G, as this glycoprotein makes it easy to recover and concentrate the pseudotyped vectors [62].
We also showed that eGFP was produced in all colony subtypes. Clusters of eGFP production were observed on fluorescence microscopy, indicating that not all the cells of a given positive colony -theoretically derived from a single cell -produced eGFP. This result is consistent with those of Mikkola et al. concerning murine HSC transduction by a VSVg-pseudotyped lentiviral vector, in which a mismatch was reported between the transduction rate of cells (almost 25%) and the transduction rate of myeloid colonies (almost 60%). These authors highlighted the occurrence of mosaicism in GFP gene expression in colonies obtained following the myeloid differentiation of CD34 + cells [63], possibly due to a delay in the integration of the transgene during differentiation, resulting in the formation of clusters of GFP-positive cells within a single myeloid colony.
In our in vivo study, autologous HSC were injected into the bone marrow, whereas intravenous injection is currently the most frequently used transplantation method. We aimed to increase seeding efficiency and homing, as only a limited number of stem cells were theoretically available. However, 2.5 × 10 6 to 5.0 × 10 6 CD34 + cells is generally sufficient to ensure engraftment, and we found that less than 2.0 × 10 6 cells were sufficient for long-term reconstitution in macaques. As predicted [64,65], totalbody gamma irradiation leads to a drastic decrease in the number of hematopoietic progenitors, preventing the development of mature cells [66]. Despite the occurrence of severe pancytopenia, a positive correlation has been found between the number of CD34 + cells infused and time required for immune reconstitution [42,67,68]. However, hematopoietic recovery may take longer if fewer than 2.0 × 10 6 CD34 + cells/kg are infused. This notion is consistent with our observation that CD34 + cell transplantation decreases both the severity and duration of irradiation-induced cytopenia. Clonogenic activity also reappeared more strongly in transplanted animals. We also showed that the animals recovered B cells, T cells, monocytes and granulocytes. Nevertheless, the functional activity of these cells requires confirmation, particularly for lymphocytes. However, although we observed long-term reconstitution with lentiviral vector-transduced cells of different lineages, its proportion remained below 1%. Hanawa et al., provided the first evidence that SIV-based vectors can successfully transduce rhesus macaque repopulating hematopoietic stem cells, with an average of 16% of peripheral blood leukocytes containing the SIV vector genome. However, this study was carried out with HSC from mobilized peripheral blood cells, making it possible to obtain larger numbers of HSC than can be harvested from bone marrow. Nevertheless theoretically, these cells contained more progenitors that were already committed and fewer pluripotent stem cells capable of long-term reconstitution than medullary HSC [69]. The small numbers of eGFP-producing cells observed in our study may be due to an anti-eGFP immune response. Some reports have suggested that such reactions do not generally occur after irradiation [70], but two reports described the induction of cytotoxic T-lymphocyte responses to enhanced green (GFP) or yellow (YFP) fluorescent proteins after myeloablative conditioning. One of these reports concerned baboons that had received primitive hematopoietic cells transduced with HIV-1-based lentiviral vectors [71] and the other concerned rhesus macaques that had received CD34 + stem cells transduced with a retroviral vector [72].
Lentiviruses, like retroviruses, can be used to integrate transgenes into the host genome. Two severe adverse events occurred in two patients in the SCID-X1 gene therapy trial 30 to 34 months after injection of the autologous CD34 + cells corrected using a retroviral vector. In these patients, an uncontrolled clonal T lymphoproliferative syndrome, similar to acute lymphoblastic leukemia, was observed [73,74]. This study highlights the risk of insertional mutagenesis restricted to retroviral and lentiviral gene transfer. In the future, additional safety measures could be considered, such as the use of self-inactivating LTRs (as in our study) to reduce enhancer activity, the addition of insulators to reduce the risk further, and the insertion of a second transgene encoding a "suicide" product, such as herpes thymidine kinase, making it possible to kill the transduced cells with ganciclovir. Unlike studies in mice, in which the follow-up period is necessarily limited, studies in large animals, with a longer life span, are compatible with more extensive follow-up. The development of linear amplification-mediated PCR (LAM-PCR), a sensitive and robust approach to molecular clonal analysis, has made it possible to identify and analyze the contribution of individual transduced clones to hematopoiesis. Clonal analysis may provide information about the dominance of transduced clones, potentially predicting possible progression or the propensity to develop clonal hematopoiesis and leukemia. Moreover, replication-competent retrovirus (RCRs), recombinant retrovirus and interaction with endogenous retroviruses should also be investigated, when evaluating the biosafety of retrovirus and lentivirus.

Conclusion
The results reported here provide the first evidence that gene transfer into medullary hematopoietic stem cells and long-term expression of the transgene are possible, using an SIV-based lentiviral vector in non human primates, which provide the best clinical models for in vivo evaluation of the feasibility and safety of gene therapy strategies.