Wiskott-Aldrich Syndrome (WAS) is an inherited immunodeficiency caused by mutations in the gene encoding WASP, a protein regulating the cytoskeleton. Hematopoietic stem/progenitor cell (HSPC) transplants can be curative but, when matched donors are unavailable, infusion of autologous HSPCs modified ex vivo by gene therapy is an alternative approach. We used a lentiviral vector encoding functional WASP to genetically correct HSPCs from three WAS patients and re-infused the cells after reduced-intensity conditioning regimen. All three patients showed stable engraftment of WASP-expressing cells and improvements in platelet counts, immune functions, and clinical score. Vector integration analyses revealed highly polyclonal and multi-lineage haematopoiesis resulting from the gene corrected HSPCs. Lentiviral gene therapy did not induce selection of integrations near oncogenes and no aberrant clonal expansion was observed after 20–32 months. Although extended clinical observation is required to establish long-term safety, lentiviral gene therapy represents a promising treatment for WAS.

There was an error published in Development 138, 3647-3656.The panel labels on the left indicating genotypes were misaligned in Fig. 5A. The corrected Fig. 5 appears in full below.The authors apologise to readers for this mistake.

The early stages of lymphoid cell formation were studied by testing the differentiative potential of phenotypically defined subsets of CD34+ bone marrow cells. A subpopulation of CD34+ Lin- CD45RA+ cells expressing CD10 was isolated by flow cytometry. Such cells are CD38+, HLA-DR+, do not express significant levels of Thy-1 and c-kit, lack erythroid, myeloid, megakaryocytic potential, and give rise only to lymphoid T, B, natural killer (NK), and dendritic cells (DC) in kinetics and titration experiments. Limiting dilution analysis demonstrates the existence of multipotential B/NK/DC progenitor clones in the CD34hi Lin-CD10+ adult bone marrow cell population. Thus, nonprimitive progenitors for lymphoid cells and for DCs can be distinct from those of myeloid, megakaryocytic, and erythroid cells, implying that the DC lineage is developmentally more closely related to the lymphoid lineage than to the myeloid lineage. This study provides new insights into the organization and development of the human lympho-hematopoietic system.

Gene transfer vectors may cause clonal imbalance and even malignant cell transformation by insertional upregulation of proto-oncogenes. Lentiviral vectors (LV) with their preferred integration in transcribed genes are considered less genotoxic than gammaretroviral vectors (GV) with their preference for integration next to transcriptional start sites and regulatory gene regions. Using a sensitive cell culture assay and a series of self-inactivating (SIN) vectors, we found that the lentiviral insertion pattern was approximately threefold less likely than the gammaretroviral to trigger transformation of primary hematopoietic cells. However, lentivirally induced mutants also showed robust replating, in line with the selection for common insertion sites (CIS) in the first intron of the Evi1 proto-oncogene. This potent proto-oncogene thus represents a CIS for both GV and LV, despite major differences in their integration mechanisms. Altering the vectors' enhancer-promoter elements had a greater effect on safety than the retroviral insertion pattern. Clinical grade LV expressing the Wiskott-Aldrich syndrome (WAS) protein under control of its own promoter had no transforming potential. Mechanistic studies support the conclusion that enhancer-mediated gene activation is the major cause for insertional transformation of hematopoietic cells, opening rational strategies for risk prevention.

Mature dendritic cells (mDCs) can trigger the effector functions of natural killer (NK) cells. Knockout , small-interfering RNA or neutralizing antibodies targeting interleukin 12 (IL-12) subunits revealed a critical role for IL-12 in NK cell interferon (IFN-) secretion promoted by mDCs. However, NK cell activation by DCs also required direct cell-to-cell contacts. DC-mediated NK cell activation involved the formation of stimulatory synapses between DCs and NK cells. The formation of DC/NK cell conjugates depended on cy-toskeleton remodeling and lipid raft mobilization in DCs. Moreover, the disruption of the DC cytoskeleton using pharmaco-logic agents or the loss-of-function mutation of the Wiskott-Aldrich syndrome protein abolished the DC-mediated NK cell activation. Synapse formation promoted the polarized secretion of preassembled stores of IL-12 by DCs toward the NK cell. The synaptic delivery of IL-12 by DCs was required for IFN-secretion by NK cells, as assessed using inhibitors of cytoskel-eton rearrangements and transwell experiments. Therefore, the cross-talk between DCs and NK cells is dictated by functional synapses. (Blood. 2004;104:3267-3275) Introduction Natural killer (NK) cells recognize and kill target cells expressing virus-encoded proteins, as well as tumor cells that have lost the expression of major histocompatibility complex (MHC) class I antigens. 1-5 Activation of NK cells results from a balance between inhibitory and activating signaling pathways. 6 Incompatibilities in HLA-Cw alleles between NK and target cells promote the cytolytic function of NK cells involved in the graft-versus-leukemia reaction. 7 In contrast, receptor-ligand interactions between MHC class I molecules and killer inhibitory immunoglobulin-like receptor (KIR) or lectin-type inhibitory NK cell receptor can initiate a dominant inhibitory signaling cascade that blocks NK cell cytotoxicity. Recent studies of the physical interaction between NK cells and target cells have highlighted the functional impact of its synaptic organization. Thus, Lou et al 8 reported that, within the NK/ target cell synapse, lipid rafts polarized to the site of the cell contact in conjugates with sensitive MHC class I-negative targets but not in conjugates with resistant MHC class I-positive targets. Moreover, the negative signals between an NK cell and a target cell are transmitted by KIR at the site of membrane apposition, where inhibitory receptors become clustered with MHC class I ligands in a supramolecular structure known as an inhibitory NK immune synapse (IS). 9,10 KIR signaling is critical for blocking lipid raft polarization and NK cell cytotoxicity, both depending on movements of microtubuli and actin filaments. 11 The composition of adhesion, costimulatory, cytoskel-etal, and signaling molecules in the supramolecular activation clusters (SMACs) of the cytolytic and noncytolytic NK cell IS revealed profound differences. 12 Indeed, cytoskeleton remodel-ing and redistribution of NK cell signaling molecules occur mainly in cytolytic NK ...

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There was an error published in Development 138, 3647-3656.The panel labels on the left indicating genotypes were misaligned in Fig. 5A. The corrected Fig. 5 appears in full below.The authors apologise to readers for this mistake.

From the perspective of a pilot clinical gene therapy trial for Wiskott-Aldrich syndrome (WAS), we implemented a process to produce a lentiviral vector under good manufacturing practices (GMP). The process is based on the transient transfection of 293T cells in Cell Factory stacks, scaled up to harvest 50 liters of viral stock per batch, followed by purification of the vesicular stomatitis virus glycoprotein-pseudotyped particles through several membrane-based and chromatographic steps. The process leads to a 200-fold volume concentration and an approximately 3-log reduction in protein and DNA contaminants. An average yield of 13% of infectious particles was obtained in six full-scale preparations. The final product contained low levels of contaminants such as simian virus 40 large T antigen or E1A sequences originating from producer cells. Titers as high as 2 × 10(9) infectious particles per milliliter were obtained, generating up to 6 × 10(11) infectious particles per batch. The purified WAS vector was biologically active, efficiently expressing the genetic insert in WAS protein-deficient B cell lines and transducing CD34(+) cells. The vector introduced 0.3-1 vector copy per cell on average in CD34(+) cells when used at the concentration of 10(8) infectious particles per milliliter, which is comparable to preclinical preparations. There was no evidence of cellular toxicity. These results show the implementation of large-scale GMP production, purification, and control of advanced HIV-1-derived lentiviral technology. Results obtained with the WAS vector provide the initial manufacturing and quality control benchmarking that should be helpful to further development and clinical applications.