Lentiviral gene transfer and ex vivo expansion of human primitive stem cells capable of primary, secondary, and tertiary multilineage repopulation in NOD/SCID mice

Piacibello, Wanda; Bruno, Stefania; Sanavio, Fiorella; Droetto, Sara; Gunetti, Monica; Ailles, Laurie; Sio, Francesca R. Santoni de; Viale, Andrea; Gammaitoni, Loretta; Lombardo, Angelo; Naldini, Luigi; Aglietta, Massimo

doi:10.1182/blood.v100.13.4391

Cited by 80 publications

(65 citation statements)

References 35 publications

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“…At least 20% of the CFCs regenerated in mice that had been repopulated with multiple clones of human cord blood cells exposed to concentrated preparations of this virus (0.5 × 10 9 to 1 × 10 9 infectious units/ml) had the vector transgene, and values of more than 50% were noted in several mice. These findings indicate that the β A-T87Q -globin lentivirus can be used to transduce human cord blood cells with long-term NOD/ SCID mouse-repopulating activity with the same efficiency as has been reported for simpler constructs (25,27,28,31,32,48,53).…”

Section: Discussionsupporting

confidence: 65%

High-level β-globin expression and preferred intragenic integration after lentiviral transduction of human cord blood stem cells

Imren¹,

Fabry²,

Westerman³

et al. 2004

J. Clin. Invest.

106

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Transplantation of genetically corrected autologous hematopoietic stem cells is an attractive approach for the cure of sickle-cell disease and β-thalassemia. Here, we infected human cord blood cells with a self-inactivating lentiviral vector encoding an anti-sickling β A-T87Q -globin transgene and analyzed the transduced progeny produced over a 6-month period after transplantation of the infected cells directly into sublethally irradiated NOD/LtSz-scid/scid mice. Approximately half of the human erythroid and myeloid progenitors regenerated in the mice containing the transgene, and erythroid cells derived in vitro from these in vivo-regenerated cells produced high levels of β A-T87Q -globin protein. Linker-mediated PCR analysis identified multiple transgenepositive clones in all mice analyzed with 2.1 ± 0.1 integrated proviral copies per cell. Genomic sequencing of vector-containing fragments showed that 86% of the proviral inserts had occurred within genes, including several genes implicated in human leukemia. These findings indicate effective transduction of very primitive human cord blood cells with a candidate therapeutic lentiviral vector resulting in the long-term and robust, erythroidspecific production of therapeutically relevant levels of β-globin protein. However, the frequency of proviral integration within genes that regulate hematopoiesis points to a need for additional safety modifications.

show abstract

Section: Discussionsupporting

confidence: 65%

High-level β-globin expression and preferred intragenic integration after lentiviral transduction of human cord blood stem cells

Imren¹,

Fabry²,

Westerman³

et al. 2004

J. Clin. Invest.

106

View full text Add to dashboard Cite

show abstract

“…the safety and efficiency of the lentiviral vector transduction system, which did not alter the biological characteristics of the cells or survival time of the animal models, caused substantial expression of genes over 20 weeks (28)(29)(30)). In the current study, using the Livin specific shrNA expression vector for gene knock-down, we successfully achieved stable suppression of Livin expression in A549 cells.…”

Section: Discussionmentioning

confidence: 99%

Silencing of Livin inhibits tumorigenesis and metastasis via VEGF and MMPs pathway in lung cancer

Lin

et al. 2015

International Journal of Oncology

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Abstract. Livin, an inhibitor of apoptosis protein (IAP), is overexpressed in various cancers and decreases tumor sensitivity to chemotherapy and radiotherapy. However, the effect of Livin on lung adenocarcinoma metastasis and the specific mechanism involved remain unclear. RNAi technology was used to stably silence Livin in A549 cells in the present study. The effect of Livin on tumor growth and invasion was investigated in lung cancer cells in vitro and animal models were established to determine the anti-metastasis ability of Livin silencing in vivo. The results indicated that Livin knock-down suppressed cell proliferation and inhibited cell invasion, accompanied by downregulation of VEGF and MMP-2/-9. Silencing of Livin resulted in the prevention of xenograft tumor formation. Seventy-five immunodeficient male BALB/C nude mice were randomly divided into three groups, the relative ratio of the areas with pulmonary nodules in the experimental group decreased from 46.71±7.27% to 11.07±2.94% compared with the negative control group (P<0.001), indicating the interaction between Livin, VEGF and MMPs. The xenograft tumor model of intravenous injection of tumor cells were successfully established and applied for the analysis of lung cancer tumorigenesis and metastasis in a time-dependent manner for the first time. Based on the reliable and reproducible animal model, our findings indicate that knock-down of Livin inhibits cell growth and invasion through blockade of the VEGF and MMPs pathways in lung cancer cells in vitro, and inhibits tumorigenesis and metastasis of lung cancer in vivo, suggesting that Livin is a promising antitumor target.

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“…24 Furthermore, vectors with a similar backbone are known to efficiently transduce human primitive hematopoietic progenitor cells such as CD34+CD38-cells or SCID-repopulating cells. 25 Thus, HIV-derived lentiviral vectors appear to be promising candidates for stem cellbased ex vivo gene therapy. The higher concentration of vector that we used provided on average two vector integrations per blood cell in mice (Figure 2d) and three vector copies in BM-DC (cells tested in Table 1).…”

Section: Discussionmentioning

confidence: 99%

A lentiviral vector encoding the human Wiskott–Aldrich syndrome protein corrects immune and cytoskeletal defects in WASP knockout mice

et al. 2004

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Wiskott-Aldrich syndrome (WAS) is an immune deficiency with thrombopenia resulting from mutations in the WASP gene. This gene normally encodes the Wiskott-Aldrich syndrome protein (WASP), a major cytoskeletal regulator expressed in hematopoietic cells. Gene therapy is a promising option for the treatment of WAS, requiring that clinically applicable WASP gene transfer vectors demonstrate efficacy in preclinical studies. Here, we describe a selfinactivating HIV-1-derived lentiviral vector encoding human WASP and show that it effectively transduced bone marrow progenitor cells of WASP knockout (WKO) mice. Transplantation of these transduced cells into lethally irradiated WKO recipients led to stable expression of WASP and correction of immune, inflammatory and cytoskeletal defects. Splenic T-cell proliferation was restored, podosomes were reinstated on bone-marrow-derived dendritic cells and colon inflammation was reduced. This shows for the first time (a) that cytoskeletal defects can be corrected in WKO mice, (b) that human WASP is biologically active in mice and (c) that a lentiviral vector is effective to express human WASP in vivo over several months. These data support further development of such lentiviral vectors for the gene therapy of WAS. Gene Therapy (2005) 12, 597-606.

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Lentiviral gene transfer and ex vivo expansion of human primitive stem cells capable of primary, secondary, and tertiary multilineage repopulation in NOD/SCID mice

Cited by 80 publications

References 35 publications

High-level β-globin expression and preferred intragenic integration after lentiviral transduction of human cord blood stem cells

High-level β-globin expression and preferred intragenic integration after lentiviral transduction of human cord blood stem cells

Silencing of Livin inhibits tumorigenesis and metastasis via VEGF and MMPs pathway in lung cancer

A lentiviral vector encoding the human Wiskott–Aldrich syndrome protein corrects immune and cytoskeletal defects in WASP knockout mice

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