Stable RNA interference: comparison of U6 and H1 promoters in endothelial cells and in mouse brain

Mäkinen, Petri; Koponen, Jonna; Kärkkäinen, Anna‐Mari; Malm, Tarja; Pulkkinen, Kati; Koistinaho, Jari; Turunen, Mikko P.; Ylä‐Herttuala, Seppo

doi:10.1002/jgm.860

Cited by 119 publications

(90 citation statements)

References 39 publications

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“…A scrambled shRNA sequence (5'-TTCTCCGAACGTGTCACGT-3') served as NC. Vectors were made as previously described [15,16]. e titers averaged to 1×10 9 TU (transduction units)/ml.…”

Section: Lp-pla 2 Rnai Lentiviral Vectorsmentioning

confidence: 99%

Amelioration of atherosclerosis in apolipoprotein E-deficient mice by inhibition of lipoprotein-associated phospholipase A2

Zhang

Zhang²,

Sun³

et al. 2013

CIM

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Purpose: Lipoprotein-associated phospholipase A2 (Lp-PLA2) is involved in the pathogenesis of atherosclerosis, especially in advanced plaques. In the present study, the abilities of darapladib, a selective Lp-PLA2 inhibitor, and lentivirus-mediated Lp-PLA2 silencing on in ammation and atherosclerosis in apolipoprotein E-de cient mice were compared.Methods: Apolipoprotein E-de cient mice were fed on a high-fat diet and a constrictive collar was placed around the le carotid artery to induce plaque formation. e mice were randomly divided into control, negative control (NC), darapladib and RNA interference (RNAi) groups. Eight weeks a er surgery, lentivirus-mediated RNAi construct or darapladib were used to decrease the expression of Lp-PLA2. Plaques were collected ve weeks later for histological analysis. In ammatory gene expression in the atherosclerotic lesions were then determined at the mRNA and protein level.Results: e expression of pro-in ammatory cytokines was signi cantly reduced in the treatment group, compared to nontreatment group, whereas the plasma concentration of anti-in ammatory cytokines increased markedly. Moreover, our results demonstrated a signi cant reduction in plaque lipid content, as well as a rise in collagen content following Lp-PLA2 inhibition. Interestingly, when comparing the two methods of Lp-PLA2 inhibition, animals treated with Lp-PLA2 RNAi were found to exhibit lower plaque areas and enhanced improvement of plaque stability as compared with animals treated with darapladib. Darapladib had no attenuating e ect on atherosclerotic plaque area. ese therapeutic e ects were independent of plasma lipoprotein levels.Conclusions: Lp-PLA2 inhibition by darapladib or lentivirus-mediated RNAi ameliorated in ammation and atherosclerosis in apolipoprotein E-de cient mice. e e ect was more prominent in the RNAi group.

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“…A scrambled shRNA sequence (5'-TTCTCCGAACGTGTCACGT-3') served as NC. Vectors were made as previously described [15,16]. e titers averaged to 1×10 9 TU (transduction units)/ml.…”

Section: Lp-pla 2 Rnai Lentiviral Vectorsmentioning

confidence: 99%

Amelioration of atherosclerosis in apolipoprotein E-deficient mice by inhibition of lipoprotein-associated phospholipase A2

Zhang

Zhang²,

Sun³

et al. 2013

CIM

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“…ARE elements, minimal SV40 promoter and the luciferase gene were digested with KpnI and XbaI and blunt ligated into the lentiviral backbone. 42 NF-kB-luc-reporter construct was cloned by SphI-HindIII digesting NF-kB elements from pNF-kB-Luc (Stratagene, La Jolla, CA, USA), and blunt ligating into the XhoI site of pGL3 promoter and then cloning NF-kB response elements, minimal SV40 promoter and luciferase gene into the lentiviral backbone as previously described. Also a control lentiviral vector containing only minimal SV40 promoter and luciferase gene was cloned.…”

Section: Lentiviral Productionmentioning

confidence: 99%

Oxidative stress-inducible lentiviral vectors for gene therapy

Hurttila

Kansanen

et al. 2008

Gene Ther

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Oxidative stress is important in several pathologies, including cardiovascular diseases such as atherosclerosis and cardiac ischemia-reperfusion injury. An important mechanism for adaptation to oxidative stress is induction of genes through the antioxidant response element (ARE), which regulates the expression of antioxidant and cytoprotective genes via the transcription factor Nrf2 (nuclear factor E2-related factor 2). As Nrf2-regulated genes are induced during oxidant stress occurring, for example, in reperfusion after ischemia, we took a novel approach to exploit ARE for the development of oxidative stress-inducible gene therapy vectors. To this end, one, two or three ARE-containing regions from human NAD(P)H:quinone oxidoreductase-1, glutamate-cysteine ligase modifier subunit and mouse heme oxygenase-1 were cloned into a vector expressing luciferase under a minimal SV40 promoter. The construct, which was the most responsive to ARE-inducing agents, was chosen for further studies in which a lentiviral vector was produced for an efficient transfer to endothelial cells. Heme oxygenase-1 (HO-1), which has well-characterized anti-inflammatory properties, was used as the therapeutic transgene. In human endothelial cells, AREdriven HO-1 overexpression inhibited nuclear factor-kB activation and subsequent vascular cell adhesion molecule-1 expression induced by tumor necrosis factor-a. We conclude that the ARE element is a promising alternative for the development of oxidative stress-inducible gene therapy vectors.

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“…14 A possible explanation for this difference in genetic stability is the higher activity of the U6 promoter, which could more effectively interfere with virus replication. 34 Deletions in RCR vectors had been observed previously to occur between direct and inverted repeats during reverse transcription. 24 However, this mode of deletion is unlikely here as no repeats were detectable around the deletion site.…”

Section: Discussionmentioning

confidence: 96%

RNAi-mediated gene silencing in tumour tissue using replication-competent retroviral vectors

et al. 2011

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RNAi represents a powerful technology to specifically downregulate the expression of target genes. For cancer research and therapy, an efficient in vivo delivery system is supposed to distribute RNAi to all tumour cells upon systemic administration. We present replication-competent murine leukaemia virus (MLV) vectors, which deliver RNAi to tumour tissue upon tail vein injection. In HT1080 cells stably expressing GFP or luciferase, GFP expression was suppressed by more than 80% and luciferase (luc) activity by more than 90%, even when only 0.1% of the cells were initially infected with reporter gene specific vectors. To demonstrate its potential, PLK1-and MMP14-specific small hairpin RNA expression cassettes were applied in the system. Upon infection, PLK1 and MMP14 levels were reduced on mRNA and protein level. MLV-shPLK1-infected cells were arrested in the G2-phase and underwent apoptosis. MLV-shMMP14-infected cells showed reduced MMP2 activity, as well as substantially reduced invasion and tumour growth. In vivo, MLV-shLuc silenced luc expression in HT1080-luc tumour tissue by more than 80% and MLV-shPLK1 reduced tumour growth substantially, demonstrating the therapeutic relevance of this system. This RNAi vector system allows long-term downregulation of target gene expression as well as efficient delivery to and distribution throughout tumour tissue in vivo.

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Stable RNA interference: comparison of U6 and H1 promoters in endothelial cells and in mouse brain

Cited by 119 publications

References 39 publications

Amelioration of atherosclerosis in apolipoprotein E-deficient mice by inhibition of lipoprotein-associated phospholipase A2

Amelioration of atherosclerosis in apolipoprotein E-deficient mice by inhibition of lipoprotein-associated phospholipase A2

Oxidative stress-inducible lentiviral vectors for gene therapy

RNAi-mediated gene silencing in tumour tissue using replication-competent retroviral vectors

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