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Gene therapy for hemophilia A would be facilitated by development of smaller expression cassettes encoding factor VIII (FVIII), which demonstrate improved biosynthesis and/or enhanced biologic properties. B domain deleted (BDD) FVIII retains full procoagulant function and is expressed at higher levels than wild-type FVIII. However, a partial BDD FVIII, leaving an N-terminal 226 amino acid stretch (N6), increases in vitro secretion of FVIII tenfold compared with BDD-FVIII. In this study, we tested various BDD constructs in the context of either wild-type or codon-optimized cDNA sequences expressed under control of the strong, ubiquitous Spleen Focus Forming Virus promoter within a self-inactivating HIV-based lentiviral vector. Transduced 293T cells in vitro demonstrated detectable FVIII activity. Hemophilic mice treated with lentiviral vectors showed expression of FVIII activity and phenotypic correction sustained over 250 days. Importantly, codon-optimized constructs achieved an unprecedented 29- to 44-fold increase in expression, yielding more than 200% normal human FVIII levels. Addition of B domain sequences to BDD-FVIII did not significantly increase in vivo expression. These significant findings demonstrate that shorter FVIII constructs that can be more easily accommodated in viral vectors can result in increased therapeutic efficacy and may deliver effective gene therapy for hemophilia A.

Ubiquitously acting chromatin opening elements (UCOEs) consist of methylation-free CpG islands encompassing dual divergently transcribed promoters of housekeeping genes that have been shown to confer resistance to transcriptional silencing and to produce consistent and stable transgene expression in tissue culture systems. To develop improved strategies for hematopoietic cell gene therapy, we have assessed the potential of the novel human HNRPA2B1-CBX3 UCOE (A2UCOE) within the context of a self-inactivating (SIN) lentiviral vector. Unlike viral promoters, the enhancer-less A2UCOE gave rise to populations of cells that expressed a reporter transgene at a highly reproducible level. The efficiency of expression per vector genome was also markedly increased in vivo compared with vectors incorporating either spleen focus-forming virus (SFFV) or cytomegalovirus (CMV) promoters, suggesting a relative resistance to silencing. Furthermore, an A2UCOE-IL2RG vector fully restored the IL-2 signaling pathway within IL2RG-deficient human cells in vitro and successfully rescued the X-linked severe combined immunodeficiency (SCID-X1) phenotype in a mouse model of this disease. These data indicate that the A2UCOE displays highly reliable transcriptional activity within a lentiviral vector, largely overcoming insertion-site position effects and giving rise to therapeutically relevant levels of gene expression. These properties are achieved in the absence of classic enhancer activity and therefore may confer a high safety profile.

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.

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.

Background: X-linked severe combined immunodeficiency (SCID-X1) is caused by mutations in the common cytokine-receptor gamma chain (gamma(c)), resulting in disruption of development of T lymphocytes and natural-killer cells. B-lymphocyte function is also intrinsically compromised. Allogeneic bone-marrow transplantation is successful if HLA-matched family donors are available, but HLA-mismatched procedures are associated with substantial morbidity and mortality. We investigated the application of somatic gene therapy by use of a gibbon-ape-leukaemia-virus pseudotyped gammaretroviral vector., Methods: Four children with SCID-X1 were enrolled. Autologous CD34-positive haemopoietic bone-marrow stem cells were transduced ex vivo and returned to the patients without preceding cytoreductive chemotherapy. The patients were monitored for integration and expression of the gamma(c) vector and for functional immunological recovery., Findings: All patients have shown substantial improvements in clinical and immunological features, and prophylactic medication could be withdrawn in two. No serious adverse events have been recorded. T cells responded normally to mitogenic and antigenic stimuli, and the T-cell-receptor (TCR) repertoire was highly diverse. Where assessable, humoral immunity, in terms of antibody production, was also restored and associated with increasing rates of somatic mutation in immunoglobulin genes., Interpretation: Gene therapy for SCID-X1 is a highly effective strategy for restoration of functional cellular and humoral immunity.

Cytotoxic T cells (CTLs) and natural killer cells play a major role in the immune response to Epstein-Barr virus (EBV) infection. In X-linked lymphoproliferative (XLP) disease, a severe immunodeficiency, immunodysregulatory phenomena are observed following EBV infection, suggesting that defects exist in these effector populations. The gene defective in XLP is SAP (signaling lymphocytic activation molecule [SLAM]-associated protein), an adaptor protein that mediates signals through SLAM and other immunoglobulin superfamily receptors including 2B4. We generated EBV-specific T-cell lines from controls and XLP patients and examined CTL function in response to different stimuli. We show that XLP patients can generate EBV-T-cell lines that are phenotypically similar to those from controls. XLP patient EBV-T-cell lines showed a significant decrease in interferon-gamma (IFN-gamma) production in response to 2B4 and autologous EBV-transformed lymphoblastoid cell line (LCL) stimulation but not in response to SLAM. Furthermore, XLP EBV-T-cell lines demonstrated markedly decreased cytotoxic activity against autologous LCLs. By retroviral gene transfer of the SAP gene into XLP EBV-T-cell lines, we show reconstitution of IFN-gamma production and of cytotoxic activity confirming SAP-dependent defects. These studies demonstrate that in XLP the lack of SAP affects specific signaling pathways resulting in severe disruption of CTL function.

Development of T and natural killer (NK) cells is critically dependent on cytokine signaling, and defects in cytokine receptor complex subunits have been shown to result in severe combined immunodeficiency (SCID) syndromes in humans and in murine models. An infant boy had typical clinical features of SCID and was found to lack NK cells in his peripheral circulation. Molecular analysis did not reveal abnormalities in his gammac or JAK-3 genes, and he was investigated for defects in the interleukin-15 (IL-15) receptor complex because functional IL-15 signaling is essential for NK cell development. Expression of the IL-2R/IL-15Rbeta chain was significantly reduced in the patient's peripheral blood mononuclear cells (PBMCs) by immunoblot, flow cytometry, and Northern blot analysis. Furthermore, IL-2 stimulation of PBMCs showed only minimal tyrosine phosphorylation of JAK-3. These data demonstrate that defects in IL-2R/1L-15Rbeta expression can lead to a unique NK-deficient SCID immunophenotype. (Blood. 2001;98:877-879)

Objective: The aim of this study was to compare the hematologic compositions of fetal blood and liver and to phenotypically quantify the hematopoietic stem and progenitor cells during early human gestation., Study Design: Fifty fetal blood samples and 50 fetal livers were collected at 10 to 17 weeks' gestation and 8 to 17 weeks' gestation, respectively. Investigations included fetal blood cell counts, determinations of red blood cell index values, and flow cytometric analyses of mononuclear cells., Results: Fetal red blood cell, white blood cell, and platelet counts all increased with gestation, reflecting hematologic development. The proportion of normoblasts decreased dramatically with gestation. Individual mature red blood cells were larger and contained more hemoglobin during early gestation. Circulating and hepatic T lymphocytes increased in number shortly before the 13th week of gestation, which reflected thymic maturation. As a proportion fetal liver contained fewer T lymphocytes than did fetal blood (2.5% vs 18.6%; P =.003) but more CD34(+) hematopoietic stem and progenitor cells (17.5% vs 4.3%; P =. 004). As a proportion, fetal liver contained more of the primitive CD34(+) and CD38(-) hematopoietic stem and progenitor cells than did fetal blood (32% vs 17%; P =.04)., Conclusion: Both fetal blood and liver provide a rich source of hematopoietic stem and progenitor cells. Fetal liver provides a richer source of more primitive hematopoietic stem and progenitor cells than does fetal blood. For stem cell transplantation we suggest that fetal livers be collected before the 13th week of gestation, because T lymphocytes are present in much greater numbers in the fetal liver after this stage of gestation. Further, we suggest that in utero stem cell transplantations in fetuses with normal immune development should be performed before the 13th week of gestation.

In the mammal, definitive hematopoietic stem cells (HSCs) are first derived from mesodermal cells within a region of the embryonic para-aortic splanchnopleura known as the aorta-gonad-mesonephros (AGM). Within this region, HSCs are thought to arise from hemangioblast precursors located in the ventral wall of the dorsal aorta. However, the factors that regulate HSC development in vivo are still largely unknown. Bone morphogenetic protein (BMP)-4, a member of the transforming growth factor beta (TGF-beta) superfamily of growth factors, is a potent ventralizing factor and has been implicated in the commitment of embryonic mesodermal cells to a hematopoietic fate in a number of systems. In the human AGM, we find that BMP-4 is expressed at high levels, and with striking polarity, in a region of densely packed cells underlying intra-aortic hematopoietic clusters. In contrast, TGF-beta1 is expressed predominantly by hematopoietic cells within the clusters. These findings implicate both BMP-4 and TGF-beta1 in the initiation and regulation of hematopoiesis in the human AGM. Furthermore, the distribution of BMP-4 expression is highly suggestive of a direct role in the specification of human hematopoietic cells from embryonic mesoderm in vivo. (Blood. 2000;96:1591-1593)

Interactions between the Wiskott-Aldrich (WAS) protein (WASp), small GTPases, and the cytoskeletal organizing complex Arp2/3 appear to be critical for the transduction of signals from the cell membrane to the actin cytoskeleton in hematopoietic cells. This study shows that Fcgamma-receptor (FcgammaR)-mediated phagocytosis is impaired in WASp-deficient peripheral blood monocytes, and that in macrophages, formation of the actin cup and local recruitment of tyrosine phosphorylated proteins is markedly attenuated. Results also show that, in normal macrophages, WASp itself is actively recruited to the cup, suggesting that assembly of this specialized cytoskeletal structure is dependent on its expression. (Blood. 2000;95:2943-2946)

Wiskott-Aldrich Syndrome protein (WASP) is the product of the gene mutated in children with Wiskott-Aldrich Syndrome (WAS). It is a predominantly cytoplasmic protein, expressed only in haematopoietic cells. It binds in vivo to the adaptor proteins Nck and Grb2, to the cytoplasmic protein-tyrosine kinase Fyn and to the small Rho-like GTPase Cdc42, which is required for formation of filopodia in fibroblasts and macrophages. WASP also interacts, directly or indirectly, with the actin cytoskeleton. Together with studies of a closely related, ubiquitously expressed protein named N-WASP, these findings suggest that WASP is a component of signalling pathways that control reorganisation of the actin cytoskeleton in haematopoietic cells in response to external stimuli. In support of this idea, haematopoietic cells from WAS patients show defects in cytoskeletal organisation that compromise their ability to polarise and to migrate in response to physiological stimuli. These defects could account for many of the clinical features of WAS. WAS is now a candidate for gene therapy based on the delivery of a wild-type WASP gene to autologous haematopoietic stem cells. In addition, recent studies of cell defects in WAS patients suggest that it may prove possible, in time, to rescue WAS cells using more conventional drug therapies.

The primary immunodeficiencies are attractive candidates for the development of gene therapy approaches based on the transduction of hematopoietic cells. We have constructed a high-titer recombinant retrovirus for expression of gp91-phox, deficiencies of which cause the X-linked form of chronic granulomatous disease (X-CGD). We have used this vector to transduce human bone marrow, using either unfractionated mononuclear cells or purified CD34+ cells as targets and evaluated several infection protocols. Efficient gene transfer to progenitors and long-term culture-initiating cells (LTC-IC) was obtained for each target population. Importantly for potential clinical application, this could be achieved without the use of exogenous cytokines or polybrene. Progenitors representing each of the lineages detectable in vitro were transduced at equal efficiencies. The vector was shown partially to restore gp91-phox deficiency and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity in transduced cells derived from X-CGD patients. These data demonstrate that it is possible to transduce primitive human hematopoietic cells efficiently and reconstitute NADPH oxidase.

Chronic granulomatous disease (CGD) comprises a heterogeneous group of inherited conditions characterized biochemically by disordered function of a unique multicomponent enzyme system present in phagocytic cells, the NADPH-oxidase. Clinically, it is characterized by recurrent bacterial and fungal infections that are relatively resistant to treatment by conventional means. Curative bone marrow transplantation has been successfully achieved in a small number of cases, but the wider application of this procedure is limited by availability of suitable donor material. Somatic gene therapy would overcome this problem, and several groups have now shown correction of the biochemical defect in hematopoietic cells by retrovirus-mediated gene transfer. However, the failure of the current generation of retroviral vectors to efficiently transduce quiescent cells greatly restricts their potential for gene transfer to pluripotent hematopoietic stem cells. Given these limitations, we have constructed vectors based on adeno-associated virus and used these to transfer a functional copy of the p47phox gene to immortalized B cells derived from patients with p47phox-deficient autosomal recessive CGD. We show stable expression of protein and restoration of NADPH-oxidase function in these cells in the absence of selection. Adeno-associated virus vectors may overcome some of the limitations of retroviral gene delivery systems and may therefore be a useful vehicle for curative gene therapy of CGD and other primary immunodeficiencies.

For somatic gene therapy to become a realistic therapeutic strategy for chronic granulomatous disease (CGD), we have to be able to assign the molecular lesion to a specific component of the NADPH oxidase and to confirm that transfer of a functional copy of the corresponding defective gene will result in correction of the cellular defect. We used an adenovirus vector expressing p47phox to transduce monocytes from patients with CGD. We showed by nitroblue-tetrazolium staining that NADPH-oxidase activity was restored to these cells. This technique offers a rapid means for molecular diagnosis. In the short term, this approach may have therapeutic potential.

The interleukin-2 (IL-2) receptor gamma-chain is a common component of several members of the cytokine receptor superfamily including those for IL-2, IL-4, IL-7, IL-9, IL-15, and possibly IL-13, and has recently been renamed the common gamma-chain (gamma c-chain). Transfection experiments have shown that the gamma c-chain participates in signal transduction by IL-2, IL-4 and IL-7, but a functional role for the gamma c-chain in biological responses by normal T cells and B cells to these cytokines has not been established. In this study, we have used X-linked severe combined immunodeficiency (X-SCID) as a naturally occurring gamma c-chain gene disruption model to examine the role of the gamma c-chain in human B-cell responses to IL-2, IL-4, IL-13, and IL-15. Our experiments show that B cells from two X-SCID patients with characterized gamma c-chain gene mutations do not respond to IL-2 or IL-15, but respond as well or better than normal B cells to both IL-4 and IL-13 in assays for B-cell activation, proliferation, and IgE secretion. This finding raises important questions about the function of the gamma c-chain in receptors for IL-4 and IL-13, and the nature of the immune defect in X-SCID.

Chronic granulomatous disease (CGD) results from defects in the phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, central to which is the membrane-bound cytochrome b-245. The cytochrome is composed of two protein subunits, the larger (gp91-phox) being deficient in X-linked CGD. In this study, we have analyzed expression of the cytochrome subunits in B-cell lines from two autosomal CGD patients for whom the disease is caused by deficiency of p22-phox, the smaller subunit. We report the presence of a 65-kD precursor of gp91-phox in the membrane fraction of both p22-phox-deficient cell lines, corresponding to the core protein with N-linked carbohydrate side chains in the high mannose form. Expression of p22-phox in these cells resulted in functional correction of NADPH oxidase. In addition, gp91-phox in the reconstituted cells was processed to its terminally glycosylated form. These data suggest that the association of the 65-kD gp91-phox precursor with p22-phox is a prerequisite for processing of the carbohydrate side chains to the complex form in the Golgi. The detection of this precursor will enable characterization of mutations disrupting the subunit interaction (either naturally occurring or derived by in vitro mutagenesis) and so aid in structure-function analysis of cytochrome b-245. Reconstitution of p22-phox-deficient cells shows the potential of gene therapy for this autosomal form of CGD.

We report the development of a relatively quick and simple method for the assessment of X inactivation status for carrier determination in families affected by X-linked agammaglobulinemia (XLA). This method utilises an immunomagnetic separation technique for B cell purification and a polymerase chain reaction (PCR) based assay for the determination of methylation status at the androgen receptor (AR) gene locus to assess whether X inactivation is random or non-random at this locus. We report the results we have obtained using this assay to investigate females known to be carriers of various X-linked immunodeficiency disorders. In addition, we investigated four females from different families affected by XLA, two of whom were of unknown carrier status, and we discuss the results obtained with this and other X-inactivation assays. A similar assay has recently been described by Allen et al. (1992) and applied to members of one family affected by XLA.

Chronic granulomatous disease (CGD) is an inherited immunodeficiency resulting from the inability of an individual's phagocytes to produce superoxide anions because of defective NADPH oxidase. The disease may be treated by bone marrow transplantation and as such is a candidate for somatic gene therapy. Two thirds of patients have defects in an X-linked gene (X-CGD) encoding gp91-phox, the large subunit of the membrane cytochrome b-245 component of NADPH oxidase. Epstein-Barr virus-transformed B-cell lines from patients with CGD provide a model system for the disease. We have used retrovirus-mediated expression of gp91-phox to reconstitute functionally NADPH oxidase activity in B-cell lines from three unrelated patients with X-CGD. The protein is glycosylated and membrane associated, and the reconstituted oxidase is appropriately activated via protein kinase C. The kinetics of superoxide production by such reconstituted cells is similar to that of normal B-cell lines. These data show the potential of gene therapy for this disease.

We have compared assays for products of the neutrophil respiratory burst in normal EBV-transformed B cell lines stimulated with agonists of protein kinase C. Those measuring O2- directly or its immediate product, H2O2, were successful. Of these, the most sensitive were the lucigenin- and luminol-based chemiluminescence assays for O2- and H2O2 respectively. Cell lines from CGD patients, with X-linked or autosomal recessive genetic defects in the neutrophil NADPH oxidase, did not respond in these assays, indicative of their inability to produce O2-. The defects in the lines studied encompass both proteins forming the cytochrome b-245 membrane component, and the 47 kDa cytosolic component of the NADPH oxidase. The possession of the disease associated phenotype by these cell lines provides evidence that in the normal situation both neutrophils and B cells produce O2- via the same system.

The molecular basis of X-linked chronic granulomatous disease (X-CGD) has recently been elucidated and the defective gene identified and isolated. Two restriction fragment-length polymorphisms have been identified using the X-CGD cDNA probe. We have analyzed eight families with X-CGD and seven normal, unrelated females and have demonstrated that these polymorphisms are not in linkage disequilibrium. This should increase to approximately 50% the proportion of families to whom first-trimester prenatal diagnosis can be offered. Unambiguous determination of carrier status in related females in informative families will also be possible. In addition, we have identified an apparently unique small deletion in the X-CGD gene in a family affected by this disease, members of which are not informative for either polymorphism. This will allow prenatal diagnosis and carrier determination in this family.