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Epstein-Barr virus (EBV)-associated posttransplant lymphoma (PTLD) is a major cause of morbidity/mortality after hematopoietic stem cell (SCT) or solid organ (SOT) transplant. Adoptive immunotherapy with EBV-specific cytotoxic lymphocytes (CTLs), although effective in SCT, is less successful after SOT where lifelong immunosuppression therapy is necessary. We have genetically engineered EBV-CTLs to render them resistant to calcineurin (CN) inhibitor FK506 through retroviral transfer of a calcineurin A mutant (CNA12). Here we examined whether or not FK506-resistant EBV-CTLs control EBV-driven tumor progression in the presence of immunosuppression in a xenogeneic mouse model. NOD/SCID/IL2rγ(null) mice bearing human B-cell lymphoma were injected with autologous CTLs transduced with either CNA12 or eGFP in the presence/absence of FK506. Adoptive transfer of autologous CNA12-CTLs induced dramatic lymphoma regression despite the presence of FK506, whereas eGFP-CTLs did not. CNA12-CTLs persisted longer, homed to the tumor, and expanded more than eGFP-CTLs in mice treated with FK506. Mice receiving CNA12-CTLs and treated with FK506 survived significantly longer than control-treated animals. Our results demonstrate that CNA12-CTL induce regression of EBV-associated tumors in vivo despite ongoing immunosuppression. Clinical application of this novel approach may enhance the efficacy of adoptive transfer of EBV-CTL in SOT patients developing PTLD without the need for reduction in immunosuppressive therapy.

X-linked lymphoproliferative disease (XLP1) arises from mutations in the gene encoding SLAM-associated protein (SAP) and leads to abnormalities of NKT-cell development, NK-cell cytotoxicity, and T-dependent humoral function. Curative treatment is limited to allogeneic hematopoietic stem cell (HSC) transplantation. We tested whether HSC gene therapy could correct the multilineage defects seen in SAP(-/-) mice. SAP(-/-) murine HSCs were transduced with lentiviral vectors containing either SAP or reporter gene before transplantation into irradiated recipients. NKT-cell development was significantly higher and NK-cell cytotoxicity restored to wild-type levels in mice receiving the SAP vector in comparison to control mice. Baseline immunoglobulin levels were significantly increased and T-dependent humoral responses to NP-CGG, including germinal center formation, were restored in SAP-transduced mice.We demonstrate for the first time that HSC gene transfer corrects the cellular and humoral defects in SAP(-/-) mice providing proof of concept for gene therapy in XLP1.

The pathophysiology of microthrombocytopenia in the Wiskott-Aldrich syndrome (WAS) and its milder form, X-linked thrombocytopenia (XLT), is unclear. Although quantitative defects are correctable by splenectomy, residual platelet abnormalities are suggestive of intrinsic disturbances of production. In contrast to human patients, murine models of WASp deficiency exhibit only mild thrombocytopenia, and platelets are of normal size. Here, we have identified a critical role for WASp during murine platelet biogenesis. By electron microscopy, WASp-deficient MKs appeared to have shed platelets ectopically within the bone marrow space. WASp-deficient megakaryocytes (MKs) also displayed defects in response to fibrillar collagen I (CI) in vitro, the major matrix component of bone. These included a loss of normal CI receptor (α2β1 integrin)-mediated inhibition of proplatelet formation, a marked abrogation of SDF-1-induced chemotactic migration of CD41+ MKs adherent to CI, and an almost complete lack of actin-rich podosomes, normally induced by interaction between CI and its receptors GPVI or α2β1 integrin. These findings highlight the central and highly specialized role of WASp in MKs during platelet biogenesis, and may provide a mechanism for the mild thrombocytopenia observed in WASp-deficient mice. In addition, they suggest a novel explanation for some of the platelet abnormalities characteristic of patients with WAS. (Blood. 2006;108:134-140)

Regulated migration and spatial localization of dendritic cells (DCs) are critical events during the initiation of physiologic immune responses and maintenance of tolerance. Here we have used cells deficient in the Wiskott-Aldrich syndrome protein (WASp) to demonstrate the importance of dynamic remodeling of the actin cytoskeleton for these trafficking processes to occur in vitro and in vivo. On fibronectin-coated surfaces, WASp-null immature murine DCs exhibited defects both of attachment and detachment, resulting in impaired net translocation compared with normal cells. The chemokinetic response to CCL21, which is critical for normal lymphatic trafficking, was also abrogated in the absence of WASp. In vivo in both fluorescein isothiocyanate (FITC) and oxazolone contact hypersensitivity models, WASp-null Langerhans cell (LC) migration was compromised, as judged by exit from the skin as well as by homing to the draining lymph node (LN). Furthermore, following systemic challenge with lipopolysaccharide (LPS) or toxoplasma-derived antigen, WASp-null DCs showed incomplete redistribution to T-cell areas in the spleen. Instead, they were retained ectopically in the marginal zone. DC trafficking in vivo is therefore dependent on a normally regulated actin cytoskeleton, which performs an essential function during maintenance of physiologic immunity and when disturbed may contribute significantly to the immunopathology of Wiskott-Aldrich Syndrome.

Megakaryocytes give rise to platelets via extension of proplatelet arms, which are released through the vascular sinusoids into the bloodstream. Megakaryocytes and their precursors undergo varying interactions with the extracellular environment in the bone marrow during their maturation and positioning in the vascular niche. We demonstrate that podosomes are abundant in primary murine megakaryocytes adherent on multiple extracellular matrix substrates, including native basement membrane. Megakaryocyte podosome lifetime and density, but not podosome size, are dependent on the type of matrix, with podosome lifetime dramatically increased on collagen fibers compared with fibrinogen. Podosome stability and dynamics depend on actin cytoskeletal dynamics but not matrix metalloproteases. However, podosomes degrade matrix and appear to be important for megakaryocytes to extend protrusions across a native basement membrane. We thus demonstrate for the first time a fundamental requirement for podosomes in megakaryocyte process extension across a basement membrane, and our results suggest that podosomes may have a role in proplatelet arm extension or penetration of basement membrane.

Introduction Human hematopoietic stem cell transplantation (HSCT) has been used to treat a range of hematological and immunological disorders. As a result, the demand for hematopoietic stem cells (HSC) in clinical applications is increasing. Amniotic fluid stem cells (AFSC) serve as a potential alternative cell source for therapy. Amniotic fluid can be derived by amniocentesis or therapeutic amniodrainage. AFSC are multi-potent, have low risk of tumorigenicity, can be expanded and do not have legal or ethical limitations. The significant hematopoietic activity of murine AFSC led us to explore the potential of human AFSC (CD117/c-Kit+) towards hematopoietic differentiation and to reconstitution in vivo. Methods Human AFSC (2nd and 3rd trimester) and cord blood HSC (CB-HSC; control) were selected for CD117 and CD34 respectively using a MoFlo XDP sorter. Sorted cells (104 in 200μl PBS) were injected intravenously into sub-lethally irradiated NOD-SCID/IL2rγnull (NSG) mice (n=6/group). Hematopoietic engraftment of human cells (% of human CD45+ within total CD45+) and multi-lineage reconstitution (myeloid: CD13, CD14, CD15 and lymphoid: CD3, CD4 and CD8) were assessed at 16 weeks in blood, bone marrow (BM) and spleen by flow cytometry. For subsequent secondary transplants, BM mononuclear cells (MNC) derived from BM harvested from primary recipients of mice were intravenously injected into secondary recipients (1.5x107 MNC in 200μl PBS). Hematopoietic engraftment was assessed at 16 weeks post-transplantation (n=6/group). For further analysis of human donor cell engraftment, Q-PCR was performed on spleen samples harvested from primary and secondary recipients using oligonucleotide primers specific for human ALU repeat sequences; Immunohistochemistry was carried out using anti-human CD45 antibody and detected with a commercially available kit (Dako EnVision Plus, Dako). Results are expressed as mean±SEM, and statistical analysis was performed using 1-way ANOVA with Bonferroni post-hoc tests. Results Human AFSC engrafted the hematopoietic system of NSG mice at levels similar to the ones achieved with CB-HSC (blood: AFSC 7.5±1.3% vs. CB-HSC 6.1±2.2%, p=0.6; BM: AFSC 46.3±7.9% vs. CB-HSC 38.3±8.2%, p=0.6; spleen: AFSC 39.6±9.3% vs. CB-HSC 34.7±10.5%, p=0.7). Similarly, at 16 weeks following secondary transplantation, human donor cell engraftment was comparable between groups in blood (AFSC 11.5 ± 3.9% vs. CB-HSC 16.9 ± 3.9%, p=0.3) and other hematopoietic tissues. Q-PCR and immunohistochemistry confirmed donor cell engraftment in AFSC and CB-HSC groups. Importantly, there were no differences between groups in multi-lineage differentiation at 16 weeks post primary and secondary transplantation. Conclusion Human CD117/c-Kit+ AFSC have functional, multi-lineage hematopoietic potential that is similar to the current "gold-standard" stem cell source for hematopoietic transplantation. The ease of isolation during early gestation, as well as their gene-engineering and expansion potential make human AFSC a novel autologous fetal cell source for pre- and post-natal therapy of inherited hematological disorders. Disclosures No relevant conflicts of interest to declare.

Severe congenital neutropenia (SCN) is characterized by neutropenia, recurrent bacterial infections, and maturation arrest in the bone marrow. Although many cases have mutations in the ELA2 gene encoding neutrophil elastase, a significant proportion remain undefined at a molecular level. A mutation (Leu270Pro) in the gene encoding the Wiskott-Aldrich syndrome protein (WASp) resulting in an X-linked SCN kindred has been reported. We therefore screened the WAS gene in 14 young SCN males with wild-type ELA2 and identified 2 with novel mutations, one who presented with myelodysplasia (Ile294Thr) and the other with classic SCN (Ser270Pro). Both patients had defects of immunologic function including a generalized reduction of lymphoid and natural killer cell numbers, reduced lymphocyte proliferation, and abrogated phagocyte activity. In vitro culture of bone marrow progenitors demonstrated a profound reduction in neutrophil production and increased levels of apoptosis, consistent with an intrinsic disturbance of normal myeloid differentiation as the cause of the neutropenia. Both mutations resulted in increased WASp activity and produced marked abnormalities of cytoskeletal structure and dynamics. Furthermore, these results also suggest a novel cause of myelodysplasia and that male children with myelodysplasia and disturbance of immunologic function should be screened for such mutations.

No defects related to deficiency of the Wiskott-Aldrich Syndrome protein (WASp) have been described in osteoclasts. Here we show that there are significant morphologic and functional abnormalities. WASp-null cells spread over a much larger surface area and are highly polykaryotic. In their migratory phase, normal cells assemble clusters of podosomes behind their leading edges, whereas during the bone resorptive phase multiple podosomes are densely aggregated in well-defined actin rings forming the sealing zone. In comparison, WASp-null osteoclasts in either phase are markedly depleted of podosomes. On bone surfaces, this results in a failure to form actin rings at sealing zones. Complementation of WASp-null osteoclasts with an enhanced green fluorescent protein (eGFP)-WASp fusion protein restores normal cytoarchitecture. These structural disturbances translate into abnormal patterns of bone resorption both in vitro on bone slices and in vivo. Although physiologic steady-state levels of bone resorption are maintained, a major impairment is observed when WASp-null animals are exposed to a resorptive challenge. Our results provide clear evidence that WASp is a critical component of podosomes in osteoclasts and indicate a nonredundant role for WASp in the dynamic organization of these actin structures during bone resorption. (Blood. 2004;103:3552-3561)

The reduced engraftment potential of hematopoietic stem/progenitor cells (HSPCs) after exposure to cytokines may be related to the impaired homing ability of actively cycling cells. We tested this hypothesis by quantifying the short-term homing of human adult CD34+ cells in nonobese diabetic/severe combined immunodeficient (NOD/SCID) animals. We show that the loss of engraftment ability of cytokine-activated CD34+ cells is associated with a reduction in homing of colony-forming cells (CFCs) to bone marrow (BM) at 24 hours after transplantation (from median 2.8% [range, 1.9%-6.1%] to 0.3% [0.0%-0.7%]; n = 3; P < .01), coincident with an increase in CFC accumulation in the lungs (P < .01). Impaired BM homing of cytokine-activated cells was not restored by using sorted cells in G0G1 or by inducing cell cycle arrest at the G1/S border. Blocking Fas ligation in vivo did not increase the BM homing of cultured cells. Finally, we tested cytokine combinations or culture conditions previously reported to restore the engraftment of cultured cells but did not find that any of these was able to reverse the changes in homing behavior of cytokine-exposed cells. We suggest that these changes in homing and, as a consequence, engraftment result from the increased migratory capacity of infused activated cells, leading to the loss of selectivity of the homing process.

Background Beta thalassaemia is a genetic blood disease that causes life-threatening anemia. Hematopoietic stem cell (HSC) transplantation successfully cures the disease but in only 30% of patients. We hypothesized that in utero gene therapy (IUGT) to the fetal HSC compartment with the corrected beta globin gene might cure the disease before birth. Methods A humanized mouse model of thalassemia (Cooley's anemia; CA) was used in which heterozygous animals are affected by anemia, splenomegaly and extra-medullary hematopoiesis. At E13.5 a “GLOBE” vector (HIV-2 based lentiviral vector that incorporates a mini hemoglobin beta gene, the beta-globin promoter and HS2/3 β-LCR element) was injected into the liver of each fetus (n=12). At 12 weeks of age, recipient blood, liver, spleen and bone marrow were collected for complete blood count, blood film, as well as RNA and DNA isolation. Extra-medullary hematopoiesis was examined in the spleen and liver using flow cytometry (CD71+/Ter119+ cells) and histo-pathological analysis. Gene expression of human and mouse alpha and beta globins, as well as human gamma globin was assessed by quantitative polymerase chain reaction (qPCR). High performance liquid chromatography (HPLC) was used to quantify the presence of human beta globin in the peripheral blood of treated animals. Results are expressed as mean±SEM, and statistical analysis was performed using 1-way ANOVA with Bonferroni post-hoc tests. Experimental protocols were approved by the ethical committee on animal experimentation at University College London. Results Compared to non-injected heterozygous pups (control), IUGT increased hemoglobin levels [11.3±0.4g/dl (n=6) vs. 7.6±0.6g/dl (n=8); p Conclusions IUGT resulted in phenotypic normalization in a heterozygous humanized mouse model of CA. Increased levels of beta globin and associated down-regulation of gamma globin is consistent with a switch from fetal to adult human hemoglobin, confirming successful prenatal correction of the genetic defect. Figure A. Quantification of human beta globin mRNA using qPCR (*p Figure B. HPLC analysis of peripheral blood hemolysates of control versus IUGT treated. Figure B. HPLC analysis of peripheral blood hemolysates of control versus IUGT treated. Disclosures No relevant conflicts of interest to declare.

The cellular mechanisms that configure the cytoskeleton during migration of dendritic cells (DCs) are poorly understood. Immature DCs assemble specialized adhesion structures known as podosomes at their leading edge; these are associated with the localized recruitment of the Wiskott-Aldrich Syndrome protein (WASp) and the actin organizing actin-related protein 2/3 complex. In immature DCs lacking WASp, podosomes are absent, residual dysmorphic lamellipodia and filopodia are nonpolarized, and migration is severely compromised. Microinjection studies indicate that podosome assembly and polarization require concerted action of Cdc42, Rac, and Rho, thereby providing a link between sequential protrusive and adhesive activity. Formation of podosomes is restricted to cells with an immature phenotype, indicating a specific role for these structures during the early migratory phase. (Blood. 2001;98:1142-1149)

Background EBV associated lymphoproliferative disease (PTLD) remains a major cause of morbidity and mortality after stem cell (SCT) or solid organ (SOT) transplant. Adoptive transfer of ex vivo-derived EBV-specific cytotoxic T lymphocytes (EBV-CTL) to reconstitute immunity to the oncogenic virus EBV has been highly effective for both the prophylaxis and treatment of PTLD after SCT, where immunosuppression could be withdrawn before transfer of EBV-CTLs. In contrast, the application of this approach for the treatment of PTLD in SOT patients, although feasible, has been challenging with restricted expansion, persistence and efficacy of the adoptive transferred T cells. This difference is likely to reflect the need for the on-going immunosuppression to prevent graft-rejection post SOT which inhibits the virus-specific T cell responses. Our group has previously genetically engineered EBV-CTL to enable them to function in vitro in the presence of the calcineurin inhibitor Tacrolimus (FK506) through retroviral transfer of a calcineurin mutant (CNA12). Aim To examine the ability of genetically engineered EBV-CTLs resistant to FK506 to control EBV+ B cell lymphoma progression in vivoin a xenogeneic mouse model in the presence of FK506. Methods NOD/SCID/IL2rgnull (NSG) mice were inoculated subcutaneously with 5×106 EBV-transformed lymphoblastoid B cell lines (LCL) labelled with F-Luc to develop human EBV+ lymphoma. To evaluate in vivo antitumor activity, 5×106 CTLs retrovirally transduced with CNA12 or eGFP control CTLs were injected intravenously after 7 days in the presence or absence of FK506 (10mg/kg/day). Tumour growth was analysed using the Xenogen-IVIS system. Results Adoptive transfer of autologous CNA12 transduced CTLs induced EBV+ lymphoma regression in the presence of FK506, as assessed both by IVIS and tumour size, whereas FK506 treated mice receiving eGFP control CTLs had tumour progression (P Conclusions Our results demonstrate that CNA12 modified EBV-CTL can induce regression of EBV-associated tumours in vivo in the face of on-going immunosuppression with FK506. Clinical application of this novel approach may enhance the efficacy of adoptive transfer of EBV-CTL in SOT patients developing PTLD without the need for reduction in immunosuppressive therapy. Disclosures: No relevant conflicts of interest to declare.

Abstract 141 Short term cytokine exposure reduces the engraftment potential of haematopoietic stem and progenitor cells (HSPC). We have previously shown, in MPB CD34+ cells, that this occurs in conjunction with reduced short term homing to the BM of irradiated NOD/SCID animals, and is evident by 4 hours of culture (Ahmed at al, Blood 2004; 103:2079). Homing and engraftment of HSPC is dependent on the SDF-1/CXCR4 axis, that is negatively regulated by CD26, a cell-surface peptidase that cleaves SDF-1. Thus, blockade of CD26 with diprotin A improves engraftment of cord blood HSPC. CD26 levels are low on MPB CD34+ cells, but increase on cytokine exposure, an effect that may account for reduced homing function. We tested the effect of diprotin A treatment on the homing and engraftment of MPB CD34+ progenitors in irradiated NOD/SCID mice. Cytokine exposure (SCF, FL, IL3, IL6 for 48–72 hrs) reduced BM homing of progenitors (to 32±7% of uncultured cells, p=0.031). Treatment with Diprotin A significantly improved the homing of cultured progenitors (p=0.0002 cf non-treated cells), to the levels seen in uncultured cells (NS cf homing of uncultured cells with or without Diprotin A). Despite this increase in levels of homing to the BM, long term engraftment of cultured progenitors is not rescued by CD26 blockade, suggesting a defect beyond the initial step of homing to BM. To assess the BM attachment of transplanted cells, we used the intrabone (IB) assay, injecting cells directly into the tibiae of irradiated recipients thus bypassing the need for homing from the systemic circulation. In comparison to intravenously (IV) injected cells, IB delivery improved engraftment of both cultured and uncultured MPB CD34+ cells. At a cell dose of 106, median engraftment of uncultured cells was 13.27% (range 2.43–49.1, n=5) by IB injection compared with 1.5% (0.1–4.6, n=6) by IV delivery (p To study this attachment defect directly, we developed an ex-vivo model where CD34+ cells are incubated in long bones of irradiated, 3-week old Sprague Dawley rats after resident HSPC are removed by vigorous flushing. Irradiation, flushing protocols and cell doses were optimized to ensure sensitivity and reproducibility of the assay. Progenitor adherence was assessed by colony assays on infused and recovered attached cells, correcting for animal weight. In this model, cultured progenitors displayed reduced attachment: median number of attached progenitors, 182.1 (23–384, n=26), compared with 385.4 (49–1124, n=30) for uncultured cells (p Next, we tested the effect of cytokine exposure on ligand-specific adhesion of MPB progenitors. CD34+ cells were incubated on immobilized ligands and progenitor adhesion assessed from the clonogenic output of non-adherent cells. Progenitor adhesion to several putative niche ligands was significantly reduced following cytokine exposure. Specific adhesion of uncultured progenitors to N-cadherin was 31.8±2%, compared to 19.7±3.2% for cultured progenitors (p=0.0058). Cytokine culture also reduced specific adhesion to osteopontin and VCAM-1 (p=0.0025 and p=0.0164 respectively), but not to fibronectin, suggesting that reduced adhesive function of cultured cells is not a global defect. We conclude that whilst short term homing of cultured MPB CD34+ cells to the BM can be improved by CD26 blockade, the resulting long term engraftment defect remains. This defect is at least partly related to altered adhesive interactions of cultured cells to ligands within the BM. Disclosures: No relevant conflicts of interest to declare.

Abstract 1498 We have previously shown that a point mutation (Ile294Thr) in the Wiskott Aldrich Syndrome Protein (WASP) detected in a Wiskott Aldrich Syndrome (WAS) patient led to enhanced actin polymerising activity of WASP 1 and well as increased instability2. We also showed that macrophages from this patient displayed an increased number of actin based adhesion structures called podosomes. Additionally, podosomes in macrophages from this patient were extremely dynamic with a high rate of turnover. Based on these results we proposed that the active open conformation of WASP promotes actin polymerisation but it also induces podosome disassembly and adhesion turnover. Although the idea that the same open conformation of WASP leads to both assembly and disassembly of podosomes may be counterintuitive at first, it is possible that for termination of podosomes actin polymerisation and integrin recruitment have to be discontinued and the same constituents of growing podosomes such as active WASP may contribute to the disassembly process. We have also shown that the rapid turn-over of podosomes involves cleavage of WASP by the protease calpain, further supporting a role of WASP in both podosome formation and disassembly. However, the specific signalling mechanisms that make active WASP susceptible to cleavage by calpain leading to podosome disassembly remain unknown and need further clarification. Phosphorylation of WASP Y291 (human) or Y293 (mouse) promotes the open conformation of WASP and results in enhanced actin polymerisation. We now report that in myeloid cells, tyrosine phosphorylation of WASP negatively regulates the stability of podosomes, leading to their calpain-dependent disassembly. Additionally, we found that constitutive phosphorylation of WASP results in extensive degradation in a process that involves calpain. Our data also indicate that phosphorylation of WASP sustains the open/active conformation that promotes cleavage of WASP by calpain. Interestingly, phosphorylated WASP can bind to the WASP interacting protein (WIP) and other proteins that form a complex with WASP in podosomes such as Nck, cortactin. Taken together, our data indicate that in myeloid cells, tyrosine phosphorylation sustains the open conformation of WASP and it enhances its susceptibility to calpain-mediated cleavage preventing accumulation of actin filaments and integrin associated proteins in podosomes. This process facilitates podosome disassembly and cell translocation. These new findings support the key role of WASP as a protein that integrates actin polymerisation and cell adhesion required for mobilisation of myeloid cells during the immune response. Reference List 1. Ancliff PJ, Blundell MP, Cory GO et al. Two novel activating mutations in the Wiskott-Aldrich syndrome protein result in congenital neutropenia. Blood 2006;108:2182-2189. 2. Moulding DA, Blundell MP, Spiller DG et al. Unregulated actin polymerization by WASp causes defects of mitosis and cytokinesis in X-linked neutropenia. J Exp. Med 2007;204:2213–2224. Disclosures: No relevant conflicts of interest to declare.

To the Editor: In a recent issue of Blood, Guenechea et al[1][1]reported that ex vivo cytokine-expanded CD34+ cord blood cells retained nonobese diabetic/severe combined immunodeficient (NOD/SCID) repopulating activity of fresh cells, although there was a significant delay in the time to achieve

The WAS gene is mutated in Wiskott-Aldrich syndrome (WAS) and in X-linked thrombopenia (XLT). These diseases associate platelet defects with variable immune dysfunction, as a result of abnormal signalling and impaired cytoskeletal regulation in hematopoeitic cells. Conceivably, severe forms of WAS could be treated by gene therapy because retroviral or lentiviral-mediated WAS gene transfer restores protein expression and function in several WAS protein-deficient models. The purpose of the present study was to improve the safety and efficacy of such lentiviral vectors. Endogenous WAS promoter elements were used to restrict transgene expression to the target cell population and to provide the possibility of regulated expression in these cells. Sequences 0.5 or 1.6 kb upstream of transcription start, were operational in the transfer vector and restricted transgene expression to hematopoietic cells. Vectors utilizing either one of the WAS promoter sequences or the ubiquitously-active PGK-1, SFFV or EF1-a promoters, were compared. Equivalent levels of WAS protein were induced in lymphocytes and dendritic cells (DC), although slightly inferior mRNA levels were obtained in B cells using WAS promoters. At the functional level, all vectors restored a similar degree of proliferation and IL-2 production in T cells and equivalent numbers of podosome cytoskeletal structures in DC. The 0.5 kb or 1.6 kb-long WAS promoter sequences functioned similarly in WAS B lymphocytes or in a model of WASP-deficient T cells generated by RNA interference. No toxicity was induced by over-expression of these vectors in CD34+ cells. Altogether, the data show that lentiviral vectors with WAS promoters function efficiently in several lineages of patient cells and support further development of a hematopoietic-restricted approach for the gene therapy of WAS.