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ABSTRACT: Gene transfer into quiescent T and B cells is important for gene therapy and immunotherapy approaches. Previously, we generated lentiviral vectors (LVs) pseudotyped with Edmonston (Ed) measles virus (MV) hemagglutinin (H) and fusion (F) glycoproteins (H/F-LVs), which allowed efficient transduction of quiescent human T and B cells. However, a major obstacle in the use of H/F-LVs in vivo is that most of the human population is vaccinated against measles. As the MV humoral immune response is exclusively directed against the H protein of MV, we mutated the two dominant epitopes in H, Noose, and NE. LVs pseudotyped with these mutant H-glycoproteins escaped inactivation by monoclonal antibodies (mAbs) but were still neutralized by human serum. Consequently, we took advantage of newly emerged MV-D genotypes that were less sensitive to MV vaccination due to a different glycosylation pattern. The mutation responsible was introduced into the H/F-LVs, already mutated for Noose and NE epitopes. We found that these mutant H/F-LVs could efficiently transduce quiescent lymphocytes in the presence of high concentrations of MV antibody-positive human serum. Finally, upon incubation with total blood, mimicking the in vivo situation, the mutant H/F-LVs escaped MV antibody neutralization, where the original H/F-LVs failed. Thus, these novel H/F-LVs offer perspectives for in vivo lymphocyte-based gene therapy and immunotherapy.
Molecular Therapy 05/2012; 20(9):1699-712. · 6.87 Impact Factor
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ABSTRACT: In vivo lentiviral vector (LV)-mediated gene delivery would represent a great step forward in the field of gene therapy. Therefore, we have engineered a novel LV displaying SCF and a mutant cat endogenous retroviral glycoprotein, RDTR. These RDTR/SCF-LVs outperformed RDTR-LVs for transduction of human CD34(+) cells (hCD34(+)). For in vivo gene therapy, these novel RDTR/SCF-displaying LVs can distinguish between the target hCD34(+) cells of interest and nontarget cells. Indeed, they selectively targeted transduction to 30%-40% of the hCD34(+) cells in cord blood mononuclear cells and in the unfractionated BM of healthy and Fanconi anemia donors, resulting in the correction of CD34(+) cells in the patients. Moreover, RDTR/SCF-LVs targeted transduction to CD34(+) cells with 95-fold selectivity compared with T cells in total cord blood. Remarkably, in vivo injection of the RDTR/SCF-LVs into the BM cavity of humanized mice resulted in the highly selective transduction of candidate hCD34(+)Lin(-) HSCs. In conclusion, this new LV will facilitate HSC-based gene therapy by directly targeting these primitive cells in BM aspirates or total cord blood. Most importantly, in the future, RDTR/SCF-LVs might completely obviate ex vivo handling and simplify gene therapy for many hematopoietic defects because of their applicability to direct in vivo inoculation.
Blood 11/2011; 119(5):1139-50. · 9.90 Impact Factor
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ABSTRACT: 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.
Journal of Virology 03/2011; 85(12):5975-85. · 5.40 Impact Factor
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ABSTRACT: In vivo targeted gene delivery to hematopoietic stem cells (HSCs) would mean a big step forward in the field of gene therapy. This would imply that the risk of cell differentiation and loss of homing/-engraftment is reduced, as there is no need for purification of the target cell. In vivo gene delivery also bypasses the issue that no precise markers that permit the isolation of a primitive hHSC exist up to now. Indeed, in vivo gene transfer could target all HSCs in their stem-cell niche, including those cells that are "missed" by the purification criteria. Moreover, for the majority of diseases, there is a requirement of a minimal number of gene-corrected cells to be reinfused to allow an efficient long-term engraftment. This requisite might become a limiting factor when treating children with inherited disorders, due to the low number of bone marrow (BM) CD34(+) HSCs that can actually be isolated. These problems could be overcome by using efficient in vivo HSC-specific lentiviral vectors (LVs). Additionally, vectors for in vivo HSC transduction must be specific for the target cell, to avoid vector spreading while enhancing transduction efficiency. Of importance, a major barrier in LV transduction of HSCs is that 75% of HSCs are residing in the G0 phase of the cell cycle and are not very permissive for classical VSV-G-LV transduction. Therefore, we engineered "early-activating-cytokine (SCF or/and TPO)" displaying LVs that allowed a slight and transient stimulation of hCD34(+) cells resulting in efficient lentiviral gene transfer while preserving the "stemness" of the targeted HSCs. The selective transduction of HSCs by these vectors was demonstrated by their capacity to promote selective transduction of CD34(+) cells in in vitro-derived, long-term culture-initiating cell colonies and long-term NOD/SCID repopulating cells. A second generation of these "early-acting-cytokine"-displaying lentiviral vectors has now been developed that is fit for targeted in vivo gene delivery to hCD34(+) cells. In the method presented here, we describe the in vivo gene delivery into hCD34(+) cells by intramarrow injection of these new vectors into humanized BALB/c Rag2( null )/IL2rgc ( null ) (BALB/c RAGA) mice.
Methods in molecular biology (Clifton, N.J.) 01/2011; 737:367-90.
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ABSTRACT: Efficient gene transfer into quiescent T and B lymphocytes for gene therapy or immunotherapy purposes may allow the treatment of several genetic dysfunctions of the hematopoietic system, such as immunodeficiencies, and the development of novel therapeutic strategies for cancers and acquired diseases. Lentiviral vectors (LVs) can transduce many types of nonproliferating cells, with the exception of some particular quiescent cell types such as resting T and B cells. In T cells, completion of reverse transcription (RT), nuclear import, and subsequent integration of the vesicular stomatitis virus G protein pseudotyped LV (VSVG-LV) genome does not occur efficiently unless they are activated via the T-cell receptor (TCR) or by survival-cytokines inducing them to enter into the G(1b) phase of the cell cycle. Lentiviral transduction of B cells is another matter because even B-cell receptor-stimulation inducing proliferation is not sufficient to allow efficient VSVG-LV transduction. Recently, a new LV carrying the glycoproteins of measles virus (MV) at its surface was able to overcome vector restrictions in both quiescent T and B cells. Importantly, naive as well as memory T and B cells were efficiently transduced while no apparent activation, cell-cycle entry, or phenotypic switch were detected, which opens the door to a multitude of gene therapy and immunotherapy applications as reported here.
Molecular Therapy 10/2010; 18(10):1748-57. · 6.87 Impact Factor
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Blood 07/2010; 116(3):498-500; author reply 500. · 9.90 Impact Factor
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ABSTRACT: Up to now, no lentiviral vector (LV) tool existed to govern efficient and stable gene delivery into quiescent B lymphocytes, which hampers its application in gene therapy and immunotherapy areas. Here, we report that LVs incorporating measles virus (MV) glycoproteins, H and F, on their surface allowed transduction of 50% of quiescent B cells, which are not permissive to VSVG-LV transduction. This high transduction level correlated with B-cell SLAM expression and was not at cost of cell-cycle entry or B-cell activation. Moreover, the naive and memory phenotypes of transduced resting B cells were maintained. Importantly, H/F-LVs represent the first tool permitting stable transduction of leukemic cancer cells, B-cell chronic lymphocytic leukemia cells, blocked in G(0)/G(1) early phase of the cell cycle. Thus, H/F-LV transduction overcomes the limitations of current LVs by making B cell-based gene therapy and immunotherapy applications feasible. These new LVs will facilitate antibody production and the study of gene functions in these healthy and cancer immune cells.
Blood 09/2009; 114(15):3173-80. · 9.90 Impact Factor
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ABSTRACT: 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.
Current Gene Therapy 01/2009; 8(6):449-60. · 3.39 Impact Factor
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ABSTRACT: 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.
Blood 10/2008; 112(13):4843-52. · 9.90 Impact Factor
