Introduction

Paroxysmal nocturnal hemoglobinuria (PNH) is a severe acquired hematopoietic stem cell (HSC) disorder characterized by one or more hematopoietic clones with a somatic mutation in the X-linked phosphatidylinositol glycan-class A (PIGA) gene coding for PIGA protein^1,2,3. It represents the catalytic subunit of the N-acetylglucosaminyl transferase enzymatic complex for the first step in glycosylphosphatidylinositol (GPI) biosynthesis^4,5. This deficit leads to deficient surface expression of glycosylphosphatidylinositol-anchored proteins (GPI-APs). The absence of complement control proteins, including decay-accelerating factor (CD55) and membrane inhibitor of reactive lysis (CD59), results in intravascular lysis of the abnormal red cells^6,7,8. The clinical course of the disease is dominated by a chronic hemolytic anemia, often associated with recurrent nocturnal exacerbations, neutropenia, thrombocytopenia, and episodes of venous thrombosis⁹. Allogenic stem cell transplantation is the only therapeutic option for severe PNH¹⁰, but requires HLA-matched donors and is itself associated with a high treatment-related morbidity and mortality with graft-versus-host disease as the most frequent cause of death¹¹. Gene therapy holds great promise for severe forms of PNH because the feasibility of this approach has been recently demonstrated with oncoretroviral vectors containing PIGA cDNA¹². However, this gene therapy approach is limited by the low rate of transduction observed with oncoretroviral vectors into primary hematopoietic cells. To be successful, clinical gene therapy of PNH would require a high percentage of transduction of HSCs to limit reinfusion of uncorrected cells and a pretransplantation bone marrow (BM) conditioning regimen to eliminate resident abnormal stem cells that could have a survival advantage¹³. Lentiviral vectors could open up a new perspective for HSC gene therapy because they can transduce nondividing cells, a crucial asset in cells such as HSC. Most human immunodeficiency virus (HIV)-based vector systems have proven efficiency in transducing human HSCs^{14,15,16,17,18,19,20}. We have previously shown that lentiviral vectors greatly improved the transduction efficiency of murine HSCs, making it possible to overcome the preselection of transduced cells to cure porphyria in a mouse model of gene therapy²¹. In the present work, we have developed a new SIN lentiviral vector (TEPW) that contains the PIGA cDNA driven by the human elongation factor 1 (EF1) promoter, the central DNA flap of HIV-1²² to improve transduction efficiency of HSCs²³ and the WPRE (woodchuck hepatitis virus posttranscriptional regulatory element) cassette to achieve a high level of expression²⁴. TEPW transduction led to a complete surface expression of the GPI anchor and CD59 in PIGA-deficient cell lines. Moreover, we show that efficient gene transfer can be achieved in BM and mobilized peripheral blood (mPB) CD34⁺ cells derived from two patients with severe PNH. This expression was stable during erythroid, myeloid, and megakaryocytic liquid culture differentiation. CD59 surface cell expression was fully restored at a high percentage in long-term culture. To our knowledge, this is the first report of very efficient, stable, and long-term restoration of GPI-AP expression in CD34⁺ cells and in their progeny from patients with PNH. These findings are promising for the future treatment of PNH patients by gene therapy.

Results

TEPW allows a full, stable, and functional restoration of GPI anchor in GPI-deficient cell lines

To explore the potential of lentiviral vectors in the management of PNH, we designed an improved lentiviral vector, TEPW, containing the central DNA flap of HIV-1 and the WPRE cassette and carrying PIGA cDNA under the control of the human EF1 promoter (Fig. 1). TEPW vector titer was determined by flow cytometry based on the restoration of CD59 surface expression following transduction of GPI-deficient K562 cells. High lentiviral titers could be achieved after concentration (5.7 10⁸ transduction units (TU)/ml). A similar high titer was obtained with TEEW control vector (1.4 10⁹ TU/ml).

Figure 1.

Schematic representations of SIN lentiviral vector constructs. (A) Different transfer vector constructs. Abbreviations: PIGA cDNA, 1542-bp coding sequence of PIGA gene; cPPT, central polypurine tract; CTS, central termination sequence; eGFP, enhanced green fluorescent protein gene; gag/RRE, truncated gag region containing the rev-responsive element; hEF1, human elongation factor 1 promoter; , packaging signal; SA, splice acceptor site; SD, splice donor site; U3, U3 region deleted of TATA box and enhancer sequences; WPRE, woodchuck hepatitis posttranscriptional regulatory element. (B) Southern blot analysis of GPI-deficient K562 (K) and Jurkat (J) cell lines transduced with TEPW vector shows appropriate single proviral band with the expected size (2.2 kb).

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To evaluate whether TEPW vector would be able to restore the GPI anchor in PNH-phenotype cells, we first transduced two human GPI-deficient hematopoietic cell lines. Cells were transduced with a range of multiplicity of infection (m.o.i.) from 0.4 to 12. On day 5 following transduction, cells were stained with a monoclonal antibody (mAB) for CD59, one of the GPI-APs, and with fluorescent aerolysin, which binds directly the GPI anchor. Data from GPI-deficient K562 cells and Jurkat cells from three independent experiments are shown in Figs. 2A and 2B. The percentage of CD59⁺ cells initially increased as a direct function of the m.o.i. and the curves flattened from the m.o.i. of 4, reaching a maximum of 99.9% CD59-positive cells. No difference was observed in the transduction efficiency of the two cell lines. The results obtained with the fluorescent aerolysin staining were similar, indicating that anti-CD59–fluorescein isothiocyanate (FITC) mAB is a reliable tool to assess the amount of GPI anchor on the cytoplasmic membrane. Restoration of GPI anchor on the K562 cell membrane was stable 6 months after transduction in liquid culture (data not shown). These experiments demonstrate the restoration of PIGA function by the TEPW vector in human hematopoietic GPI-deficient cell lines. A Southern blot analysis of genomic DNA from deficient K562 and Jurkat transduced cells demonstrated the presence of a single 2.2-kb band, indicating unarranged provirus (Fig. 1B). In addition, a semiquantitative Southern blot from deficient K562 transduced at various m.o.i. shows an increasing copy number consistent with the increase in m.o.i. (Fig. 2C).

Figure 2.

Gene transfer by TEPW vector into GPI-deficient hematopoietic cell lines restores GPI anchor and overcomes resistance to aerolysin. GPI-deficient K562 (A) and Jurkat (B) cell lines were transduced with the TEPW vector at a m.o.i. of 0.4 to 12. Five days after transduction, cells were stained with anti-CD59–FITC monoclonal antibody or fluorescent aerolysin, which binds directly GPI anchor, for analysis by flow cytometry. The open bars represent the percentage of total cells expressing CD59 and the solid bars, the percentage of total cells expressing fluorescent aerolysin. (C) A semiquantitative Southern blot analysis was performed on genomic DNA from untransduced (0) and TEPW-transduced GPI-deficient K562 at various m.o.i. (0.4, 1.2, 4, and 12) as described for Fig. 1. One hundred fifty, 50, and 15 pg of TEPW plasmid were used with 14 g of normal DNA corresponding to 5, 1.5, and 0.5 copies per cell of the transgene, respectively.

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CD34⁺ hematopoietic stem and progenitor cells (HSPCs) are known to be difficult to transduce, necessitating high levels of m.o.i. Because overexpression of GPI could be a problem and to test the GPI expression level at high m.o.i., GPI-deficient K562 cells were transduced with the TEPW vector with a range of m.o.i. from 4 to 400 (Fig. 3). At the same time, cells were transduced with the TEEW control vector at the same m.o.i. to evaluate GFP level expression. GPI expression was stable in transduced cells (mean fluorescence intensities (MFI) from 36 to 60) and was similar to that in the normal K562 cell line. In contrast, the increase in m.o.i. with the TEEW vector resulted in a marked increase in GFP expression, as shown by the MFI, which increased 12-fold between the m.o.i. of 4 and of 400 (MFI from 634 to 7604). Thus, TEPW vector does not induce overexpression of GPI anchor despite high m.o.i.

Figure 3.

Lentiviral-mediated gene transfer by TEPW vector and expression into GPI-deficient K562 cell line. Transduced cells were harvested 5 days after a single round of transduction with the TEPW or TEEW vector at a m.o.i. of 4 to 400 and analyzed by FACS for CD59 (CD59–FITC), GPI (fluorescent aerolysin; FLAER), or GFP expression. The mean fluorescence intensity (MFI) of CD59⁺, GPI⁺, or GFP⁺ cells is indicated. These MFI are also indicated for the normal phenotype K562 cell line.

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Aerolysin is a toxin which binds some of the GPI-APs and GPI anchor itself, killing cells by forming a channel in their membranes. To test the sensitivity of genetically modified cells to aerolysin, TEPW-transduced cells (K562 and Jurkat) were incubated for 3 h with 10^-8 M aerolysin and stained with propidium iodide to assess their viability. Data of three independent experiments are summarized in Fig. 4. These results confirm that GPI-deficient cell lines are resistant to aerolysin. Moreover, the percentage of nonviable cells is similar to that of the CD59⁺ cell fraction, irrespective of the m.o.i. and independent of the cell line. Like CD59 expression, the percentage of nonviable cells initially rose as a direct function of the m.o.i. and the curves flattened from the m.o.i. of 4, reaching a maximum of 99.8% propidium iodide-positive cells. Therefore, TEPW vector restores cell susceptibility to aerolysin, corresponding to a functional GPI anchor on the cell membrane.

Figure 4.

Lentiviral-mediated gene transfer by TEPW vector overcomes resistance to aerolysin. Transduced GPI-deficient K562 (A) and Jurkat cells (B) were exposed to 10^-8 M aerolysin for 3 h at 37°C. Cells were stained with propidium iodide to assess viability and analyzed by flow cytometry: nonviable cells were those exhibiting fluorescence. Open bars represent the percentage of nonviable cells without aerolysin exposure and solid bars the percentage of nonviable cells after 10^-8 M aerolysin exposure. Values represent means standard errors of three independent experiments.

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Full phenotypic correction of CD34⁺ HSPCs derived from BM or mPB of patients with PNH

To investigate the efficiency of the TEPW vector in CD34⁺ HSPCs bearing PNH phenotype, we transduced CD34⁺ cells derived from BM aspirate (BM CD34⁺) or peripheral blood (mPB CD34⁺) after mobilization with G-CSF, from two patients with PNH. Cells were prestimulated overnight in a RM/CytoV1 medium and were transduced twice at 24-h interval. Five days after transduction, CD59 expression was detected in the transduced cells by flow cytometry (Fig. 5) and results are summarized in Table 1. In mPB CD34⁺-derived cells transduced at a m.o.i. of 200 and 400 2, CD59⁺ cells were measured at 80.4 and 91.5%, respectively (vs 7.7 and 7.4% in untransduced cells). The percentage of CD59⁺ cells within the CD34⁺ human cell fraction was calculated to be 83.3 and 90.5%, respectively (vs 14.7 and 11.3% for control cells). Likewise, transduction of BM CD34⁺-derived cells at a m.o.i. of 400 2 led to high transgene expression levels (81.3% CD59⁺ and 81.6% CD59⁺/CD34⁺). Furthermore, CD59 expression level in transduced cells was similar to that of normal CD34⁺ cells. Thus, lentiviral gene transfer allows full restoration of PIGA function in GPI-deficient CD34⁺ HSPCs from patients with PNH.

Figure 5.

Lentiviral-mediated gene transfer by the TEPW vector into PNH-phenotype CD34⁺ hematopoietic progenitor stem cells restores PIGA function. After 18 h of cytokine prestimulation, CD34⁺ cells were transduced twice at a m.o.i. of 200 ((A) mPB CD34⁺-derived cells) or 400 ((B) mPB CD34⁺- and (C) BM CD34⁺-derived cells). Five days after the last transduction, cells were stained with anti-CD34–PC5 (y axes) and anti-CD59–FITC (x axes) monoclonal antibodies and analyzed by flow cytometry. The percentage of cells in each quadrant is indicated.

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Table 1 - Lentiviral transduction of PNH-phenotype CD34⁺ cells with the TEPW vector.

Full table

Stability of transgene expression in clonogenic progenitor cells and during hematopoietic differentiation

To investigate the transgene expression in hematopoietic differentiated progenies, transduced PNH-phenotype CD34⁺ cells were grown in liquid cultures for 1 week, under unilineage conditions favoring erythroid (human recombinant (rh) stem cell factor (SCF), rh erythropoietin (rhEPO), and rh insulin-like growth factor 1 (rhIGF-1)), myeloid (rhSCF, rhGM-CSF, and rh interleukin-6 (rhIL-6)), or megakaryocytic (rh thrombopoietin (rhTPO), rhIL-3, and rhIL-6) differentiation. Then, cells were double-stained with FITC-conjugated anti-CD59 and phycoerythrin (PE)-conjugated anti-glycophorin A or anti-CD33 or with phycoerythrin cyanine 5 (PC5)-conjugated anti-CD41 and analyzed by FACS. Results are summarized in Table 2. Figure 6 shows a FACS analysis of differentiated liquid culture derived from mPB CD34⁺ cells transduced at a m.o.i. of 400 2. TEPW vector was expressed in the erythroid as well as the myeloid or megakaryocytic progeny of transduced mPB CD34⁺ cells (up to 90.3, 70.2, and 74.6% CD59⁺ cells within the differentiated cell fraction, respectively), while less than 8% of untransduced differentiated cells were CD59⁺. Similarly, erythroid and myeloid cells derived from BM CD34⁺ expressed CD59 highly (97.8 and 87.8%, respectively). Thus, these experiments demonstrate that PIGA cDNA transfer in GPI-deficient CD34⁺ HSPCs from patients with PNH is stable during in vitro hematopoiesis. In addition, we studied CD59 expression after 7 weeks of long-term ex vivo expansion of megakaryocytes and found percentages of CD59 expression similar to those observed in short-term culture (72.4% CD59⁺/CD41⁺ vs 6.5% CD59⁺/CD41⁺ for untransduced cells)²⁹.

Figure 6.

Restoration of PIGA function in genetically modified PNH-phenotype CD34⁺ hematopoietic progenitor stem cells is stable during hematopoietic differentiation. FACS analyses of mPB CD34⁺-derived cells transduced with the TEPW vector at a m.o.i. of 400 2, showing CD59 (x axes) expression in erythroid (A), myeloid (B), and megakaryocytic (C) cells (y axes), 12 days after the last transduction. The percentage of cells in each quadrant is indicated.

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Table 2 - Lentiviral-mediated gene transfer in hematopoietic differentiated progenies.

Full table

We also evaluated the TEPW transduction of GPI-deficient lymphocytes (Fig. 7). Freshly isolated CD34-negative BMMCs from a patient with PNH were subjected to negative selection for GPI expression with the use of 3 10^-9 M aerolysin and grown in liquid cultures for 1 week, under conditions favoring T lymphoid expansion (rhIL-2 and phytohemagglutinin A (PHA)). Then, cells were transduced once with TEPW vector at a m.o.i. of 200. After infection, cells were maintained for 5 days in the presence of rhIL-2 and PHA. CD3 and CD59 expressions were detected by flow cytometry. Thus, more than 99.9% of cells were CD3⁺, proving their T lymphoid origin, and TEPW vector allowed phenotypic correction of transduced GPI-deficient T lymphocytes (80.5% CD59⁺ vs 16% of untransduced cells).

Figure 7.

Lentiviral vector TEPW restores PIGA function in PNH-phenotype T lymphocytes. BMMCs from a PNH patient were maintained for 7 days under T lymphoid expansion culture conditions (rhIL-2 and phytohemagglutinin A) with 0.3 10^-9 M aerolysin. Then, cells were transduced with the TEPW vector at a m.o.i. of 200. Five days after the transduction, cells were stained with anti-CD3–PC5 and anti-CD59–FITC monoclonal antibodies and analyzed by flow cytometry.

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To assess whether the transgene expression level was stable, colony-forming cell (CFC) assays were performed to test the efficiency of stable gene transfer into clonogenic erythroid progenitor cells (Table 1).

After 14 days of culture in methylcellulose, individual erythroid colonies were randomly picked and scored (Fig. 8A). A similar clonogenicity of untransduced and transduced CD34⁺ cells suggested an absence of viral toxicity. Then, each colony was analyzed by FACS for glycophorin A and CD59 expression (Fig. 8B). All the colonies were glycophorin A positive, evidence of their erythroid origin. We found that 57.1% (m.o.i. = 200 2, n = 21) and 82.1% (m.o.i. = 400 2, n = 28) of total colonies derived from mPB CD34⁺ cells were CD59⁺, while no CD59⁺ colonies derived from untransduced cells could be detected. Similarly, 67% (n = 9) of total colonies derived from BM CD34⁺ cells were CD59⁺. To correlate CD59 expression and transgene integration at the level of the individual colony, the presence of PIGA cDNA sequence in each colony derived from mPB CD34⁺ cells was analyzed by PCR (Figs. 8C and 8D). This showed 71.4% (m.o.i. = 200 2) and 82.1% (m.o.i. = 400 2) of total colonies to be positive. Furthermore, almost all CD59⁺ colonies showed proviral integration (91.6 and 95.6%, respectively). Obviously, no colony derived from untransduced cells was found positive by PCR. Thus, PCR analysis on CFC-derived colonies confirmed the proportion of progenitors that had been transduced. Moreover, a high correlation between proviral integration and gene correction demonstrates a high rate of expression following proviral integration at the clonal level.

Figure 8.

Lentiviral-mediated gene transfer into colony-forming progenitor cells is stable. Five days after the last transduction, CFC assays were performed with mPB CD34⁺ cells transduced with TEPW vector at a m.o.i. of 200 or 400 2. (A) Total colonies of BFU-E, CFU-E, and CFU-GM were counted after 14 days in culture. The values were calculated for 3 10³ CD34⁺ cells plated in methylcellulose. (B) Representative FACS analyses of two individual erythroid colonies, stained with anti-glycophorin A–APC (y axes) and anti-CD59–FITC (x axes) monoclonal antibodies: PNH-phenotype colony on the left and normal phenotype on the right. (C and D) Transduction efficiency of erythroid colonies as detected by PCR analysis of individual colonies derived from mPB CD34⁺ cells transduced with the TEPW vector. (C) PCR analysis (representative examples) of randomly picked individual colonies. Abbreviations: NC, negative control; PC, positive control; NT, nontransduced colonies; PCR +, PCR-positive colony; PCR -, PCR-negative colony. (D) This table summarizes the results of immunostaining and PCR for each colony in the two experiments.

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TEPW allows full and long-term phenotypic correction of CD34⁺ HPSCs from PNH patients

Finally, to assess transduction of TEPW vector in primitive hematopoietic progenitors, transduced (m.o.i. = 200 or 400 2) mPB CD34⁺ cells were cocultured over murine BM stromal cell layer for 5 weeks. Each week, cells were analyzed for CD59 expression (Table 1 and Fig. 9).

Figure 9.

Lentiviral-mediated gene transfer by TEPW vector allows long-term phenotypic correction of PNH-phenotype mPB CD34⁺ derived cells. CD59 expression was first evaluated after transduction (day 7) by flow cytometry after immunostaining with anti-CD59–FITC monoclonal antibody. Then, long-term liquid culture was initiated on a murine adherent stromal cell layer and maintained for 5 weeks. Nonadherent cells from weekly semi-depopulations of long-term culture of TEPW-transduced cells were analyzed for CD59 expression.

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At week 5, cells were plated in methylcellulose for clonogenic assays and all individual colonies were picked for PCR analysis to test proviral integration. After a drop in CD59 expression between days 7 and 14 in both experiments, the percentage of CD59⁺-transduced cells was relatively stable around 55% (m.o.i. = 200 2) and 70% (m.o.i. = 400 2) and finally reached the same value (60.9 and 61.2%, respectively) at week 5 (vs less than 13% CD59⁺ in untransduced cells). Interestingly, the percentage of CD59^- in control cells was stable over time, without any survival advantage in vitro. A PCR analysis confirmed the presence of TEPW-encoded cDNA PIGA sequence in 58% (m.o.i. = 200 2, n = 19) of total colonies derived from liquid long-term culture CD34⁺ cells. Thus, TEPW vector allows long-term phenotypic correction of primitive hematopoietic progenitors bearing the PNH phenotype.

Discussion

Lentiviral vectors based on HIV have inherent advantages in transducing nondividing cells such as HSCs, presumably by import of the viral DNA through the nuclear pore and subsequent integration into the host genome²². The vector backbone chosen for this study contains several improvements over conventional lentiviral vectors: first, the addition of the central DNA flap of HIV-1, resulting in an increase in the transduction efficiency; second, the promoter for the human EF1, resulting in a homogeneous transgene expression in hematopoietic cells; third, the incorporation of the WPRE sequence to improve the level of expression. The incorporation of the WPRE cassette has been shown to enhance the expression of both reporter and therapeutic genes from several different promoters^19,24,25,26.

We have demonstrated that two deficient cell lines with GPI-deficient phenotype, K562 and Jurkat cells, can be efficiently transduced with the TEPW vector. A high transduction efficiency was obtained at a low m.o.i., leading to almost a full correction of cell population without any cell sorting. For purposes of safety, a prerequisite for gene therapy studies is to know the effect of overexpression of a transgene on deficient cells. To investigate this issue, we transduced the K562 deficient cell line at a very high m.o.i. Despite a very high transduction rate as shown by the GFP control vector, the level of GPI anchor expression and GPI-APs such as CD59 surface membrane remained at a stable level similar to that of normal cells. These data strongly suggest that PIGA protein is not a limiting step in the biosynthesis pathway of the GPI anchor and that it requires neither careful regulation of the transgene nor a single proviral integration.

Currently, BM and mPB CD34⁺ cells are widely used for allogenic and autologous transplantation and are considered good targets for gene therapy of immunological^27,28, hematological²⁹, and metabolic disorders^30,31. As a clinical trial for gene therapy in PNH will be envisaged in the future, an important step is to demonstrate the feasibility of high-level transduction and phenotypic correction in deficient CD34⁺ cells and their progenies. The only report of restoration of GPI-APs in deficient cells with oncoretrovirus-mediated gene transfer was published recently¹². The authors demonstrated a low level of transduction in deficient BM mononuclear cells (14%) and normal mPB CD34⁺ cells (6%). Using our TEPW lentivector, we found a high percentage of gene transfer, leading to the first genetic correction of the PNH phenotype from CD34⁺ mPB and BM cells (82–90%). PNH is an HSC disorder in which cells deficient in GPI-linked proteins are found not only within red cells but also within the monocytes, granulocytes, platelets, and lymphocytes. For this reason, it was critical to demonstrate the stability of CD59 expression following transduction of CD34⁺-deficient cells, not only in erythroid cells but also in myeloid and megakaryocytic lineages. In particular, platelet examination was performed because thrombosis is a frequent clinical feature of PNH and causes morbidity and mortality. In addition, we studied CD59 expression after 7 weeks of long-term ex vivo expansion of megakaryocytes after mPB CD34⁺ cell transduction and found a similar percentage of CD59 expression as observed in short-term culture²⁹. We complemented these studies on differentiation with CFC studies, whose main goal was to determine the correlation between proviral integration and CD59 expression at a clonal level. A high correlation between proviral integration and gene correction demonstrated that almost every proviral integration led to full correction of the clone, suggesting a very high rate of expression from this vector. These lineage studies were complemented with the transduction of deficient lymphocytes, which showed a high level of genetic correction without any selection procedure. This was an important point to check because GPI-linked proteins are required for normal lymphocyte function³². Moreover, a high level of phenotypic correction of deficient mPB cells (60%) transduced with the TEPW vector was obtained, a finding consistent with proviral integration in CFCs derived from long-term culture (58%). The major requirement for the gene therapy of PNH, i.e., to obtain a very high percentage of transduction of HSCs to avoid reinfusion of uncorrected cells, was satisfied in the present study. However, two major difficulties remain for a clinical trial to be launched: first, the presence of resident abnormal stem cells; second, the persistence of BM failure, which could lead to damage of genetically corrected cells and reexpansion of the residual PNH clone(s). The underlying mechanism driving the entire process of PNH clone expansion is still unknown but it is very likely that it is due to an autoimmune attack on normal stem cells^33,34. Therefore, a pretransplantation BM conditioning regimen prior to gene therapy would be required for PNH, with the double advantage of eliminating resident abnormal stem cells and acting as immunosuppressive therapy. Indeed, treatment with immunosuppressive therapy improves survival of patients with aplasic anemia, often associated with PNH³⁵. Finally, at this time, an efficient gene therapy approach to PNH cannot be considered without myelosuppressive conditioning prior to BMT¹².

The toxicity of complete myeloablative conditioning by chemotherapy would be undesirable, however, for a nonmalignant disease such as PNH. In addition, spontaneous long-term remission can occur in PNH⁹, a factor to be taken into account when considering potentially dangerous treatments. An initial approach to resolving the problem of engraftment would be to use nonablative conditioning. Recently, nonmyeloablative stem cell transplantation was established as a novel approach for ensuing engraftment without severe regimen-related toxicities³⁶. This successful application of allogenic nonmyeloablative transplantation for PNH holds great promise that partial myelosuppression might also be sufficient prior to stem cell gene therapy. Moreover, the safety and efficacy of low-intensity nonmyeloablative conditioning combined with stem cell gene therapy have recently been demonstrated for the treatment of ADA-SCID disease³⁷.

An improvement in the efficiency of future gene therapy in PNH could be the design of efficient selection procedures aimed at increasing the frequency of genetically corrected cells. The restoration of GPI-anchor proteins on the membrane surface of genetically corrected cells could greatly facilitate the development of preselective gene therapy strategy based on the immunoenrichment of corrected hematopoietic cells prior to BMT, without the requirement of a selectable marker²⁹. Another selective approach, which could be continued in vivo after BMT, is the use of bicistronic vectors encoding therapeutic genes and genes conferring resistance to potentially toxic drugs such as the multidrug resistance 1 gene MDR1^38,39, variants of dihydrofolate reductase^40,41, and human O⁶-methylguanine–DNA methyltransferase (MGMT)^42,43,44. It has recently been demonstrated that a limiting number of MGMT-transduced marrow progenitors can repopulate mice with a nonmyeloablative dose of after alkylating drug selection⁴⁵. This system could be applied as a dual gene therapy for many genetic diseases, including PNH disease.

We have used an improved lentiviral protocol with high concentration of cytokines and high m.o.i. leading to up to 91% of CD34⁺ corrected cells. Achieving long-term pancellular expression implies the use of high-titer vectors, leading to multiple proviral integrations^46,47. However, this situation increases the risk of insertional mutagenesis, promoting the appearance of a malignant phenotype of the cell. Unfortunately, this has been recently demonstrated in mouse⁴⁸ and human⁴⁹, leading to a new reflection on how to define this risk better ⁵⁰.

The recent development of transgenic mice with PIGA gene inactivation in the hematopoietic lineage leading to circulating hematopoietic cells with deficiency in GPI-AP holds promise for preclinical stem cell gene therapy of PNH^51,52,53. Our novel lentiviral vector now has to be evaluated in these new animal models of PNH to determine their efficacy and safety in vivo, a prerequisite of great importance for the acceptance of a gene therapy clinical trial for this severe disease.

Materials and methods

Construction of vectors

A self-inactivating lentiviral plasmid, TripU3-EF1 (hereafter called TEE), containing the green fluorescent protein (eGFP) cDNA under the control of the promoter of the human EF1, was kindly provided by P. Charneau (Institut Pasteur, Paris, France), F. Pflumio, and A. Dubart-Kupperschmitt (INSERM, Villejuif, France) and has been described²³. The PIGA cDNA was obtained by polymerase chain reaction from the plasmid pCD2-neo-PIGA³ kindly provided by T. Kinoshita (Osaka University, Osaka, Japan), as follows. The 1542-bp PIGA cDNA coding sequence with BamHI and XhoI ends was obtained using the following primers: 5'-TTGGATCCAGCATGGCCTGTAGAGGAGGAG-3', sense primer, and 5'-CTCGAGCTACCTGGTTTCAGATATCTC-3', antisense primer. The integrity of the sequence of PIGA cDNA was verified by sequencing. The eGFP sequence was replaced by a BamHI/XhoI PIGA cDNA restriction fragment using BamHI/XhoI restriction sites in the TEE plasmid. The XhoI/KpnI WPRE restriction fragment was inserted downstream of the PIGA cDNA, to yield the TEPW vector. The XhoI/KpnI WPRE restriction fragment was inserted downstream of the eGFP sequence in the TEE to give rise to the TEEW vector.

Flow cytometric analysis

Flow cytometric analysis was performed on a FACSCalibur flow cytometer (Becton–Dickinson, San Jose, CA). To test the expression of the GPI anchor, cells were stained with fluorescent aerolysin (Protox Biotech, Victoria, BC, Canada), a fluorescently labeled inactive variant of the toxin aerolysin. It binds selectively to GPI anchor and gives an accurate assessment of the amount of GPI anchor on the cytoplasmic membrane. GPI-AP expression was studied with FITC-conjugated anti-mouse antibody to CD59 (Clinisciences, Montrouge, France). To analyze lineage-specific expression, cells were stained with PE-conjugated anti-mouse antibodies to glycophorin A, CD33, and CD34; PC5-conjugated anti-mouse antibodies to CD3 (BD BioSciences, BD Pharmingen, Le Pont de Claix, France); and allophycocyanine (APC)-conjugated anti-mouse antibodies to glycophorin A (Becton–Dickinson).

Production and titration of vectors

Vector particles were produced by transient calcium phosphate cotransfection of human kidney 293T cells by the vector plasmid (10 g); an encapsidation plasmid lacking all accessory HIV-1 protein, pCMVP8.91 (10 g); and a vesicular stomatitis virus envelope expression plasmid, VSV-G pMD.G (5 g)¹⁶. Viral supernatant was collected and concentrated by ultracentrifugation. Viral titer of TEEW was determined on 293T cells with serial dilutions of viral supernatant. Expression of eGFP was determined directly by flow cytometry. TEPW viral supernatant was titered by assaying transduction of GPI-deficient K562 cells⁵⁴ (kindly provided by E. Medof, Case Western University, Cleveland, OH), with a serial dilution of viral supernatant. Expression of GPI-AP was analyzed by flow cytometry after immunolabeling with the anti-CD59 monoclonal antibody. Lentiviral preparations were tested for the absence of replication-competent lentivirus (RCL) as described¹⁶ and were found to be free of RCL. Briefly, peripheral blood mononuclear cells (PBMCs) were infected with 10⁷ or 10⁸ vector particles/ml and a quantification of HIV p24 antigen was performed by an enzyme-linked immunosorbent assay (INNOTEST HIV antigen mAb) (Innogenetics, Marseille, France) in cell culture supernatant collected over a period of 3 weeks. Controls were run in parallel, using serial dilutions of wild-type HIV-infected PBMCs.

Cell lines and transduction

The GPI-deficient K562 cells⁵⁴ and GPI-deficient Jurkat cells⁵⁵ (kindly provided by J. Schubert, Hannover Medical School, Hannover, Germany) were maintained in RPMI 1640 medium (Gibco BRL, Life Technologies, Paisley, Scotland), supplemented with 10% heat-inactivated fetal calf serum (BioWhittaker, Emerainville, France) at 37°C. Cells (10⁵) were transduced with serial dilutions of viral supernatants from a m.o.i. of 0.4 to 400, in the presence of 8 g/ml protamine sulfate (Sigma–Aldrich, Saint-Quentin-Fallavier, France), in a final volume of 0.3 ml. Controls were run in parallel, using nontransduced cells. On day 7 following transduction, cells were harvested and analyzed by FACS for GFP, CD59, and GPI expression.

Aerolysin assay

Aerolysin is a toxin secreted by Aeromonas hydrophila that induces cell death by binding to GPI-anchored receptors. Aerolysin is produced by trypsin activation of proaerolysin (Protox Biotech)⁵⁶. Cells (10⁵) were incubated for 3 h at 37°C with aerolysin (10^-8 M). Then, cells were washed and resuspended in 0.5 ml. Propidium iodide was added to each tube and the mixture was incubated for 1 h at 4°C before analysis on FACS. Nonviable cells, which exhibit sensitivity to aerolysin, incorporate propidium iodide and were therefore fluorescent.

Hematopoietic progenitor cell isolation and transduction

Human BM aspirate was obtained at the Centre Hospitalier et Universitaire de Bordeaux (France), from a patient with PNH who had given informed consent. Mobilized peripheral blood CD34⁺ cells were obtained after mobilization of progenitor/stem cells by G-CSF, with the aim of PB autologous transplantation⁵⁷. Mononuclear cells were obtained by Ficoll–Paque density centrifugation (Amersham Pharmacia Biotech, Orsay, France). Cells were washed three times with phosphate-buffered saline (PBS) containing 2 mM EDTA, and CD34⁺ cells were enriched using a Miltenyi MiniMACS CD34 Isolation Kit (Miltenyi Biotech, Paris, France). BM CD34-negative cells were harvested for T lymphoid expansion in liquid medium. CD34⁺ cells were incubated in RM/Cyto V1 medium (MABIO-International Laboratories, Tourcoing, France), a serum-free medium RM/B00 supplemented with the following human recombinant cytokines: 100 ng/ml rhSCF, 100 ng/ml rh FMS-like tyrosine kinase 3-ligand (rhFLT3-L), 100 ng/ml rhTPO, 20 ng/ml rhIL-3. CD34⁺ cells were prestimulated overnight in RM/Cyto V1 prior to viral exposure. Cells (10⁴ to 10⁵) were transduced twice at 24-h interval with TEPW vector at a m.o.i. of 200 or 400 in RM/Cyto V1 supplemented with 8 g/ml protamine sulfate. Controls were run in parallel, using nontransduced cells. Before the second transduction, cells were centrifuged and resuspended in fresh medium. CD34⁺-derived cells were harvested 5 days after the second infection for FACS analysis, clonogenic assays, and liquid culture differentiation.

T lymphoid expansion and transduction

BM CD34-negative-derived cells were placed in RM/B00 (MABIO-International), a serum-free medium supplemented with 10 ng/ml rhIL-2 (R&D Systems, Minneapolis, MN) and 5 mg/ml PHA. Cells were exposed to aerolysin at 0.3 10^-9 M, to eliminate the GPI-positive cellular fraction. After 7 days of culture, 10⁵ cells were transduced once with TEPW vector at a m.o.i. of 200. Controls were run in parallel, using nontransduced cells. T lymphocytes were harvested 5 days after transduction and analyzed by FACS for CD3 and CD59 expression.

CFC assays

Five days after the second transduction, CD34⁺-derived cells (3 10³ and 10⁴) were plated in duplicate in methylcellulose medium DM-MS02 (MABIO-International) for colony assays of hematopoietic cells. Erythroid burst-forming units (BFU-E), erythroid colony-forming units (CFU-E), and granulocyte–macrophage colony-forming units (CFU-GM) were scored 14 days after plating. BFU-E were harvested and washed twice with PBS. Each colony was analyzed for glycophorin A and CD59 expression and was screened by PCR to detect provirus integration.

Assays for primitive progenitors in long-term culture

Five days after the second transduction, 2 10⁴ to 10⁵ CD34⁺-derived cells were cocultured over the MS-5 murine BM stromal cell line in 1 ml of long-term culture medium Myelocult H5100 (Stemcell Technologies, Meylan, France), supplemented with 10 ng/ml rhSCF and 5 ng/ml rhFLT3-L. The culture was semi-depopulated each week. Cells recovered from weekly semi-depopulations were analyzed for CD59 expression by FACS. After 5 weeks, adherent and nonadherent cells, containing progenitor cells, were harvested and pooled and then plated in methylcellulose medium for clonogenic assays, as described above. Individual hematopoietic colonies were subsequently picked from plates and washed twice for further PCR analysis.

Detection of provirus integration

Integration of PIGA cDNA transgene was performed by PCR analysis on DNA extracted from CFCs or long-term culture-derived colonies. DNA was prepared as follows: cells were digested with proteinase K in lysis buffer (10 mm/L Tris–Cl, pH 8.0, 50 mmol/L KCl, 2.5 mmol/L MgCl₂, 0.5% Tween 20, 100 g/ml proteinase K) at 50°C for 1 h, followed by a 10-min incubation at 95°C. Amplification of genomic DNA was performed on the entire extract. The presence of provirus was characterized by PCR using specific primers designed in the PIGA cDNA sequence to generate a 517-bp fragment: P1, 5'-CAAACCACATCATTTGTGTG-3', as a forward primer and P2, 5'-GGCTCACATAAAATAATAAGG-3', as a reverse primer. The amplification was performed with 36 to 42 cycles of denaturation (30 s at 94°C), annealing (40 s at 54°C), and extension (20 s at 72°C). Nontransduced colonies were also used as negative controls.

Genomic DNA Southern blot was performed on transduced cell lines to test the integrity of the provirus by digestion with BamHI. Digested DNA was gel fractionated on 0.8% agarose gel, blotted, and hybridized with ³²P-labeled WPRE probe.

Myeloid, erythroid, and megakaryocytic differentiation

The differentiation culture conditions were started on day 7, after ex vivo culture and transduction. The CD34⁺-derived cells were placed under erythroid culture conditions until day 10 in RM/B00 medium supplemented with the following cytokines: 25 ng/ml rhSCF, 3 U/ml rhEPO (Eprex; Janssen-Cillag SA, Boulogne-Billancourt, France), and 50 ng/ml rhIGF-1 (R&D Systems). Then, from day 10 to day 14, cells were maintained in the same medium without rhSCF. For myeloid differentiation, cells were maintained in RM/B00 supplemented with 25 ng/ml rhSCF and 10 ng/ml rhGM-CSF (generous gift from B. Dazey, Etablissement Français du Sang Aquitaine-Limousin, Bordeaux, France). For megakaryocytic differentiation, cells were suspended in RM/B00 supplemented with 10 ng/ml rhIL-3 (generous gift from B. Dazey), 10 ng/ml rhIL-6 (CellGenix, Inc., Gaithersburg, MD), and 100 ng/ml rhTPO (CellGenix). Cultures were harvested on day 14 and analyzed for CD59 and lineage surface marker expression by FACS. In addition, a stroma-free liquid culture expansion was performed in RM/B00 supplemented with 10 ng/ml rhTPO, 50 ng/ml rhSCF, and 10 ng/ml rhIL-6 to monitor the long-term stability of megakaryocyte genetic correction⁵⁸. Every week for 6 weeks, the 1-ml culture wells were carefully resuspended and then semi-depopulated by removal of one-half the culture volume, which was replaced by fresh medium and growth factors.

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Acknowledgements

We thank Dr. P. Charneau (Institut Pasteur, Paris, France) for providing TripU3-EF1 plasmid; Dr. T. Kinoshita (Osaka University, Osaka, Japan) for providing the plasmid pCD2-neo-PIGA; Dr. J. Schubert (Hannover Medical School, Hannover, Germany) for providing GPI-deficient Jurkat cells; Dr. E. Medof (Case Western University, Cleveland, OH) for providing GPI-deficient K562 cells; Professor H. Fleury (Bordeaux, France) for RCR assays, and I. Lamrissi-Garcia for technical assistance. This work was supported by the Association pour la Recherche contre le Cancer (ARC), Conseil Régional d'Aquitaine, and Association Française contre les Myopathies (AFM).