Singer, O and Verma, IM. Applications of lentiviral vectors for shRNA delivery and transgenesis. Curr Gene Ther 8: 483-488

The Salk Institute for Biological Studies, La Jolla, CA 92037, USA.
Current Gene Therapy (Impact Factor: 2.54). 01/2009; 8(6):483-8. DOI: 10.2174/156652308786848067
Source: PubMed


Lentiviral vectors are potent gene delivery vehicles that enable stable expression of transgenes in both dividing and post-mitotic cells. Development of lentiviral vectors expressing small hairpin RNAs generates a system that can be used to down regulate specific target genes in vivo and in vitro. In this review, we will discuss two examples of in vivo applications for the use of lentiviral vectors expressing shRNAs: Gene therapy of neurological disorders and generation of transgenic knockdown animals.

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    • "In contrast, using recombinant viral vectors for gene delivery to patients with chronic disease would benefit from both cost and therapeutic efficiency . Lentiviral vectors are able to infect a wide variety of dividing and nondividing cells, integrate stably into the host chromosomes, and result in long-term expression of the transgene (Singer and Verma, 2008). Upon one or two shots, the therapeutic levels of transgene can be usually achieved. "
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    ABSTRACT: Immune cells are involved in the pathogenesis of osteoarthritis (OA). CD4+ T cells were activated during the onset of OA and induced macrophage inflammatory protein (MIP)-1γ expression and subsequent osteoclast formation. We evaluated the effects of local knockdown of MIP-1γ in a mouse OA model induced by anterior cruciate ligament-transection (ACLT). The mouse macrophage cell lines and osteoclast-like cells generated from immature hematopoietic monocyte/macrophage progenitors of murine bone marrow were co-cultured with either receptor activator of NFκB ligand (RANKL) or CD4+ T cells. The levels of MIP-1γ, RANKL in cells and mice were examined by enzyme-linked immunosorbent assay (ELISA). The osteoclastogenesis was evaluated using tartrate-resistant acid phosphatase (TRAP) and cathepsin K staining. OA was induced in one hind-leg knee joint of B6 mice. Lentiviral vector encoding MIP-1γ small hairpin RNA (shRNA) and control vector were individually injected intraarticularly into these knee joints. The knee joints were histologically assessed for manifestations of OA. The expression of MIP-1γ, MMP-13, the infiltration of CD4+ T cells, macrophages and osteoclastogenesis in tissues were examined using immunohistochemistry (IHC). CD4+ T cells were involved in osteoarthritis by inducing MIP-1γ expression in osteoclast progenitors and the subsequent osteoclast formation. Neutralizing MIP-1γ with a specific antibody abolishes RANKL- and CD4+ T cell-stimulated osteoclast formation. MIP-1γ levels were significantly higher in synovium and the chondro-osseous junction of joints 90 days post-surgery. The number of infiltrated CD4+ T cells, macrophages and IL-1β expression was reduced in the synovial tissues of mice treated with MIP-1γ shRNA. Histopathological examinations revealed that mice treated with MIP-1γ shRNA had less severe osteoarthritis than control mice had, as well as decreased osteoclast formation and matrix metalloproteinase (MMP)-13 expression. Locally inhibiting MIP-1γ expression may ameliorate disease progression and provide a new OA therapy.
    Full-text · Article · Sep 2013 · Human gene therapy
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    • "Several groups have developed vector-based siRNA expression systems that can induce RNAi in living cells. With lentiviral vectors for shRNA expression, high transduction efficiencies can be achieved, which minimizes the effects of clonal selection of phenotypically diverse cells [34]. Therefore, ADAM17 shRNA expression vectors were constructed in the present study, and ADAM17 expression was significantly inhibited by lentivirus-mediated ADAM17 RNAi in A549 cells. "
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    ABSTRACT: The purposes were to study the role of lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF)-α/nuclear factor-κB (NF-κB) signaling in matrix metalloproteinase 9 (MMP9) expression in A549 cells and to investigate the effects of lentivirus-mediated RNAi targeting of the disintegrin and metalloproteinase 17 (ADAM17) gene on LPS-induced MMP9 expression. MMP9 expression induced by LPS in A549 cells was significantly increased in a dose- and time-dependent manner (p<0.05). Pyrrolidine dithiocarbamate (PDTC) and a TNFR1 blocking peptide (TNFR1BP) significantly inhibited LPS-induced MMP9 expression in A549 cells (p<0.05). TNFR1BP significantly inhibited LPS-induced TNF-α production (p<0.05). Both PDTC and TNFR1BP significantly inhibited the phosphorylation of IκBα and expression of phosphorylation p65 protein in response to LPS (p<0.05), and the level of IκBα in the cytoplasm was significantly increased (p<0.05). Lentivirus mediated RNA interference (RNAi) significantly inhibited ADAM17 expression in A549 cells. Lentivirus-mediated RNAi targeting of ADAM17 significantly inhibited TNF-α production in the supernatants (p<0.05), whereas the level of TNF-α in the cells was increased (p<0.05). Lentiviral ADAM17 RNAi inhibited MMP9 expression, IκBα phosphorylation and the expression of phosphorylation p65 protein in response to LPS (p<0.05). PDTC significantly inhibited the expression of MMP9 and the phosphorylation of IκBα, as well as the expression of phosphorylation p65 protein in response to TNF-α (p<0.05). Lentiviral RNAi targeting of ADAM17 down-regulates LPS-induced MMP9 expression in lung epithelial cells via inhibition of TNF-α/NF-κB signaling.
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    • "Plasmid-based short hairpin RNA (shRNA) is an advanced modification of RNAi, which produces robust and prolonged RNA inhibition and knockdown of the target protein [22], [23]. Lentiviral vectors provide a means to express shRNA and induce stable and long-term gene silencing in both dividing and non-dividing cells [24]. In previous studies, we have developed a highly efficient method for lentiviral-mediated delivery of shRNAs that target PKCγ, allowing in vivo gene silencing in the spinal cord of rats and identification of novel targets of PKCã that may be involved in neuropathic pain [20], [25]. "
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    ABSTRACT: Morphine tolerance is a common drawback of chronic morphine exposure, hindering use of this drug. Studies have shown that PKCã may play a key role in the development of morphine tolerance, although the mechanisms are not fully known. In a rat model of morphine tolerance, PKCã knockdown in the spinal cord was successfully carried out using RNA interference (RNAi) with lentiviral vector-mediated short hairpin RNA of PKCã (LV-shPKCã). Spinal cords (L4-L5) were obtained surgically from morphine-tolerant (MT) rats with and without PKCã knockdown, for comparative proteomic analysis. Total proteins from the spinal cords (L4-L5) were extracted and separated using two-dimensional gel electrophoresis (2DGE); 2D gel images were analyzed with PDQuest software. Seven differential gel-spots were observed with increased spot volume, and 18 spots observed with decreased spot volume. Among these, 13 differentially expressed proteins (DEPs) were identified with matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), comparing between MT rats with and without PKCã knockdown. The DEPs identified have roles in the cytoskeleton, as neurotrophic factors, in oxidative stress, in ion metabolism, in cell signaling, and as chaperones. Three DEPs (GFAP, FSCN and GDNF) were validated with Western blot analysis, confirming the DEP data. Furthermore, using immunohistochemical analysis, we reveal for the first time that FSCN is involved in the development of morphine tolerance. These data cast light on the proteins associated with the PKCã activity during morphine tolerance, and hence may contribute to clarification of the mechanisms by which PKCã influences MT.
    Preview · Article · Jul 2012 · PLoS ONE
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