Advanced Design of Minimalistic Dumbbell-shaped Gene Expression Vectors
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Author:
Date:
2017-08-05
[Abstract] Minimal DNA vectors exclusively comprising therapeutically relevant sequences hold great promise for the development of novel therapeutic regimen. Dumbbell-shaped vectors represent non-viral non-integrating DNA minimal vectors which have entered an advanced stage of clinical development (Hardee et al., 2017). Spliceable introns and DNA nuclear import signals such as SV40 enhancer sequences are molecular features that have found multiple applications in plasmid vectors to improve transgene expression. In dumbbells however, effects triggered by introns were not investigated and DNA-based nuclear import sequences have not found applications yet, presumably because dumbbell vectors have continuously been minimized with regard to size. We investigated the effects of an intron and/or ...
[摘要] 唯一包含治疗相关序列的最小DNA载体对于新型治疗方案的发展具有很大的希望。哑铃型载体代表已经进入临床发展的晚期阶段的非病毒非整合DNA最小载体(Hardee等人,2017)。可接合的内含子和DNA核输入信号如SV40增强子序列是在质粒载体中发现多个应用以改善转基因表达的分子特征。然而,在哑铃中,由内含子引发的效应未被研究,基于DNA的核导入序列尚未发现应用,可能是因为哑铃载体在尺寸上不断被最小化。我们调查内含子和/或SV40增强子衍生序列对哑铃载体驱动的报告基因表达的影响。发现可拼接内含子的实现在所有研究的细胞系中无条件地增强基因表达。相反,SV40增强子的使用改善了细胞类型依赖性的基因表达。虽然这两个特征显着增加哑铃载体大小,但内含子和增强子或两者的组合都不会对基因表达产生负面影响。相反,与质粒或对照哑铃相比,这两个特征在一起改善了哑铃驱动的基因表达,高达160-或56倍。因此,强烈建议考虑用于哑铃矢量设计的内含子和SV40增强子。这种先进的设计可以促进哑铃型DNA载体的临床前和临床应用。 【背景】虽然许多基因已经使用哑铃形DNA载体表达,但大多数这些应用使用包括启动子,编码序列(CDS)和转录终止子的基本设计。一些载体包括嵌合内含子,然而,没有报道通过这种设计增强了转基因表达(Schirmbeck等人,2001)。在这里,我们研究了分子特征引发的效应,这些特征经常在哑铃驱动基因表达的质粒设计中得到应用:1.已知来自人β-珠蛋白基因剪接的嵌合内含子促进RNA加工,核出口,随后基因表达(Luo ...
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Murine Leukemia Virus (MLV)-based Coronavirus Spike-pseudotyped Particle Production and Infection
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Author:
Date:
2016-12-05
[Abstract] Viral pseudotyped particles (pp) are enveloped virus particles, typically derived from retroviruses or rhabdoviruses, that harbor heterologous envelope glycoproteins on their surface and a genome lacking essential genes. These synthetic viral particles are safer surrogates of native viruses and acquire the tropism and host entry pathway characteristics governed by the heterologous envelope glycoprotein used. They have proven to be very useful tools used in research with many applications, such as enabling the study of entry pathways of enveloped viruses and to generate effective gene-delivery vectors. The basis for their generation lies in the capacity of some viruses, such as murine leukemia virus (MLV), to incorporate envelope glycoproteins of other viruses into a pseudotyped virus ...
[摘要] 病毒假型颗粒(pp)是包膜病毒颗粒,通常来自逆转录病毒或弹状病毒,在其表面上携带异源性包膜糖蛋白和缺乏必需基因的基因组。这些合成的病毒颗粒是天然病毒的更安全的替代品并且获得由所使用的异源性包膜糖蛋白控制的嗜性和宿主进入途径特征。它们已被证明是用于具有许多应用的研究中非常有用的工具,例如使得能够研究包膜病毒的进入途径并产生有效的基因递送载体。它们的产生的基础在于一些病毒如鼠白血病病毒(MLV)的能力,以将其他病毒的包膜糖蛋白掺入假型病毒颗粒中。这些可以被工程化以包含报道基因例如荧光素酶,使得能够在易感细胞的假型粒子感染时定量病毒进入事件。在这里,我们详细说明使用中东呼吸综合征冠状病毒(MERS-CoV)尖峰(S)作为待并入的异源包膜糖蛋白的实例,能够产生基于MLV的假型包装颗粒的方案。我们还描述了这些颗粒如何用于感染易感细胞并通过荧光素酶测定进行定量感染性读数。 关键词:假型颗粒,鼠白血病病毒,包膜糖蛋白,冠状病毒, [背景] ...
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Reporter Assay for Semen-mediated Enhancement of HIV-1 Infection
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Author:
Date:
2016-07-20
[Abstract] Semen contains amyloid fibrils that enhance HIV-1 infection (Münch et al., 2007; Kim et al., 2010; Roan et al., 2011; Arnold et al., 2012; Usmani et al., 2014; Roan et al., 2014). Positively charged semen amyloids capture negatively charged viral particles and increase their attachment rates to the cell surface resulting in enhanced fusion and infection (Roan et al., 2009). Since semen is highly cytotoxic, we developed an assay that allows quantification of the infection enhancing activity of semen while minimizing its cell damaging activity. Here, we describe two protocols that allow the quantification of the infectivity enhancing activity of semen using a reporter cell line (TZM-bl cells) or peripheral blood mononuclear ...
[摘要] 精液含有增强HIV-1感染的淀粉样蛋白原纤维(Münch等,2007; Kim等,2010; Roan等,2011; Arnold等,2012; Usmani等,2014; Roan et al。 ,2014)。 带正电的精液淀粉样蛋白捕获带负电的病毒颗粒并增加其对细胞表面的附着率,导致增强的融合和感染(Roan等人,2009)。 由于精液具有高度的细胞毒性,我们开发了一种能够定量精液感染增强活性同时最大限度降低其细胞损伤活性的测定方法。 在这里,我们描述允许使用报道细胞系(TZM-bl细胞)或外周血单核细胞(PBMC)定量精液的感染性增强活性的两种方案。
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