A major limitation of current lentiviral vectors (LVs) is their inability to govern efficient gene transfer into quiescent cells such as primary T cells, which hampers their application for gene therapy. Here we generated high-titer LVs incorporating Edmonston measles virus (MV) glycoproteins H and F on their surface. They allowed efficient transduction through the MV receptors, SLAM and CD46, both present on blood T cells. Indeed, these H/F-displaying vectors outperformed by far VSV-G-LVs for the transduction of IL-7–prestimulated T cells. More importantly, a single exposure to these H/F-LVs allowed efficient gene transfer in quiescent T cells, which are not permissive for VSV-G-LVs that need cell-cycle entry into the G1b phase for efficient transduction. High-level transduction of resting memory (50%) and naive (11%) T cells with H/F-LVs, which seemed to occur mainly through SLAM, was not at cost of cell-cycle entry or of target T-cell activation. Finally, the naive or memory phenotypes of transduced resting T cells were maintained and no changes in cytokine profiles were detected, suggesting that T-cell populations were not skewed. Thus, H/F-LV transduction of resting T cells overcomes the limitation of current lentiviral vectors and may improve the efficacy of T cell–based gene therapy.

Key Points New LVs allow transduction of unstimulated hematopoietic stem cells.

Follicular dendritic cells (FDC) are involved in the presentation of native Ags to B cells during the secondary immune response. Some authors consider FDC to be hemopoietic cells, whereas others believe them to be mesenchymal cells. The low proportion of FDC in the lymphoid follicle, together with technical difficulties in their isolation, make these cells difficult to study. We show that Fibroblast Medium can be used successfully to isolate and maintain FDC lines. In this culture medium, we obtained 18 FDC lines from human tonsils, which proliferated for as long as 18 wk and showed a stable Ag phenotype as detected by flow cytometry and RT-PCR. FDC lines were CD45-negative and expressed Ags associated to FDC (CD21, CD23, CD35, CD40, CD73, BAFF, ICAM-1, and VCAM-1) and Ags specific for FDC (DRC-1, CNA.42, and HJ2). These cell lines were also able to bind B cells and secrete CXCL13, functional activities characteristic of FDC. Nevertheless, the additional expression of STRO-1, together with CD10, CD13, CD29, CD34, CD63, CD73, CD90, ICAM-1, VCAM-1, HLA-DR, alkaline phosphatase, and α-smooth muscle actin (α-SM actin) indicated that FDC are closely related to bone marrow stromal cell progenitors. The expression of α-SM actin also relates FDC with myofibroblasts. Like myofibroblasts, FDC lines expressed stress fibers containing α-SM actin and were able to contract collagen gels under the effect of TGFβ1 and platelet-derived growth factor. These findings suggest that FDC are a specialized form of myofibroblast and derive from bone marrow stromal cell progenitors.

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Gene transfer into quiescent T and B cells is of importance for gene therapy and immunotherapy approaches to correct hematopoietic disorders. Previously, we generated lentiviral vectors (LVs) pseudotyped with the Edmonston measles virus (MV) hemagglutinin and fusion glycoproteins (Hgps and Fgps) (H/F-LVs), which, for the first time, allowed efficient transduction of quiescent human B and T cells. These target cells express both MV entry receptors used by the vaccinal Edmonston strain, CD46 and signaling lymphocyte activation molecule (SLAM). Interestingly, LVs pseudotyped with an MV Hgp, blind for the CD46 binding site, were completely inefficient for resting-lymphocyte transduction. Similarly, SLAM-blind H mutants that recognize only CD46 as the entry receptor did not allow stable LV transduction of resting T cells. The CD46-tropic LVs accomplished vector-cell binding, fusion, entry, and reverse transcription at levels similar to those achieved by the H/F-LVs, but efficient proviral integration did not occur. Our results indicate that both CD46 and SLAM binding sites need to be present in cis in the Hgp to allow successful stable transduction of quiescent lymphocytes. Moreover, the entry mechanism utilized appears to be crucial: efficient transduction was observed only when CD46 and SLAM were correctly engaged and an entry mechanism that strongly resembles macropinocytosis was triggered. Taken together, our results suggest that although vector entry can occur through the CD46 receptor, SLAM binding and subsequent signaling are also required for efficient LV transduction of quiescent lymphocytes to occur.Measles virus (MV) belongs to the paramyxoviridae family and is the causative agent of measles disease. It has two envelope glycoproteins (gp's), the hemagglutinin (H) and fusion (F) glycoproteins (Hgp and Fgp, respectively), which mediate receptor binding and fusion, respectively (28, 29). Signaling lymphocyte activation molecule (SLAM) (CD150) is the receptor for both clinical isolates and vaccine strains (49, 55). However, vaccine strains like Edmonston (Edm) have gained, in addition to entry through the SLAM receptor, entry through the CD46 receptor after their adaptation in SLAM-negative cells (25,54). Moreover, recent findings suggest the existence of a third MV receptor in epithelial cells (54). CD46 is a complementregulatory molecule expressed on all human nucleated cells (27), whereas SLAM is constitutively expressed at the surfaces of some T and B cell subsets and upregulated upon proliferation of T and B lymphocytes and mature dendritic cells (DCs) (3,8). The cellular distribution of SLAM determines lymphoid tropism and explains in part the immunosuppressive character of measles virus.Importantly, even though wild-type and vaccine MV strains have been extensively studied at the levels of virulence (55), immunosuppression, and immune response (4, 21, 36) and the crystal structures of CD46 and SLAM receptor binding to MV hemagglutinin have recently been elucidated (7,17,41), there are still few data abo...

Vectors derived from retroviruses such as lentiviruses and onco-retroviruses are probably among the most suitable tools to achieve a long-term gene transfer since they allow stable integration of a transgene and its propagation in daughter cells. Lentiviral vectors should be preferred gene delivery vehicles over vectors derived from onco-retroviruses (MLV) since in contrast to the latter they can transduce non-proliferating target cells. Moreover, lentiviral vectors that have the capacity to deliver transgenes into specific tissues are expected to be of great value for various gene transfer approaches in vivo. Here we provide an overview of innovative approaches to upgrade lentiviral vectors for tissue or cell targeting and which have potential for in vivo gene delivery. In this overview we distinguish between three types of lentiviral vector targeting strategies (Fig 1): 1) targeting of vectors at the level of vector-cell entry through lentiviral vector surface modifications; 2) targeting at the level of transgene transcription by insertion of tissue specific promoters into lentiviral vectors; 3) a novel microRNA technology that rather than targeting the 'right' cells will 'detarget' transgene expression from non-target cells while achieving high expression in the target-cell. It is clear that each strategy is of enormous value for several gene therapy approaches but combining these three layers of transgene expression control will offer tools to really overcome several drawbacks in the field such as side-effect of off-target expression, clearance of transgene modified cells by immune response to the transgene and lack of biosecurity and efficiency in in vivo approaches.

Wiskott-Aldrich syndrome (WAS) gene therapy requires highly efficient and well-controlled vectors. Here we studied the performance of a lentiviral vector (LV) harbouring a 500-bp fragment of the WAS proximal promoter (WW), which we previously characterized as haematopoietic-specific and capable of restoring WAS phenotype in patients' T cells. We used an LV (WE) expressing eGFP to evaluate whether this promoter was following the expression pattern of endogenous WASp. Transgene expression was analysed in WE-transduced hCD34 + population and its progeny after in vitro and in vivo differentiation in the Rag 2 À/À , gc À/À humanized mouse. We revealed very poor expression from the WE internal promoter in macrophages and erythroid cells. Therefore, we designed a novel LV including a fragment of the alternative WAS promoter in WE vector (AWE). This new vector sustained high transgene levels along the whole lymphoid lineage in vivo. Most importantly, the performance of AWE vector was highly superior to WE vector since AWE clearly improved transgene levels in in vitro and in vivo hCD34 + -derived macrophages, erythroid cells, megakaryocytes and B cells while supporting a high expression in human T cells. This emphasizes that it is a suitable LV backbone for gene therapy of haematopoietic diseases such as WAS. Gene Therapy (2008) 15, 930-941;