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EBM:RD114可提供一个有效及多样的方法伪态化慢病毒载体

来源:美国科学促进会 2010-10-08 10:34

治疗性的慢病毒载体已变成分子医学上重要的工具,越来越多的临床试验都使用这种载体系统,从基础研究的观点来看,慢病毒载体是令人着迷的物质,一方面,第一型人类免疫缺陷病毒(HIV)可以提供较大的容量及能力将基因转殖到不分裂的细胞如骨髓干细胞(HSCs) 及神经细胞。但一些顾虑如可能产生会复制的病毒颗粒,使得我们必须移除HIV基因的一大部份,主要是外套膜蛋白质。在十月份《实验生物及医学》(Experimental Biology and Medicine) 期刊发表的特色文章,Bell等人发现来自内生性猫病毒RD114的外套膜蛋白质可以提供一个有效及多样的方法伪态化慢病毒载体,这工作由Anthony Bell执行,和哥伦比亚大学医学院的David Fegen、Maureen Ward、及Arthur Bank共同合作。

Bell博士说明" 大部份的慢病毒载体都是伪态的或包裹在水泡性口炎病毒(VSV-G) 的外套膜内,VSV-G让慢病毒载体可以有较广的细胞感染性,也较稳定,病毒颗粒较高的稳定性可以让病毒液经高速离心浓缩以增加感染性,但VSV-G对细胞有毒性,使得带此外套膜的伪态慢病毒载体无法持续的表达。因此我们研究不具毒性的病毒外套膜如RD114,RD114是有特别吸引力的另一种选择,因为这个外套膜也同样有高稳定性,HSCs也表达受体可以被感染。因此RD114可以被证明是治疗慢病毒载体系统的一个重要成员。

这个研究团队比较三个外套膜和二个慢病毒载体的包装效率:VSV-G、RD114、及RDpro,RDpro是一个RD114-HIV嵌合体设计为高效率的伪态慢病毒载体,在短暂的表达下,如预期的VSV-G有最高的力价,但非毒性的RD114能使得一般的转感染反应时间从48小时延长到96小时,VSV-G的转感染通常限制在48小时,因此RD114外套膜比起VSV-G提供一个有效的方法可以增加慢病毒产出的总量。RD114的多样性也经更进一步的探讨表现出来,利用稳定产出的RD114蛋白质,伪态慢病毒载体再经由反式的作用,产生" 混合的"慢病毒表达系统。

Bell博士说明"我们对于混合表达系统的方式所得到的结果很兴奋,我们也很惊讶地发现RD114蛋白质的表现比嵌合体的RDpro好。在基因治疗方面,RDpro悬浮液可以有效的转染周边血液HSCs,我们相信这程度的转染可以再经浓缩步骤优化后而增强。"

《实验生物及医学》期刊主编Steven R. Goodman说 "Anthony Bell及其同事令人兴奋的研究证明了使用不具毒性的外套膜蛋白质是伪态慢病毒载体的另一种选择,它对未来的基因治疗方式有很大的潜力。"(生物谷Bioon.com)

生物谷推荐英文摘要:

Exp. Biol. Med. 2010;235:1269-1276 doi:10.1258/ebm.2010.010053

RD114 envelope proteins provide an effective and versatile approach to pseudotype lentiviral vectors
Anthony J Bell, Jr1, David Fegen2, Maureen Ward3 and Arthur Bank3

1 Department of Chemistry and Biochemistry, The University of Southern Mississippi, Hattiesburg, MS
2 Helen Wills Neuroscience Institute, University of California at Berkeley, Berkeley, CA
3 Department of Genetics and Development, Columbia University, New York, NY, USA

Lentiviral vectors derived from the HIV-1 genome offer great promise for gene therapy due to their ability to transduce non-dividing cells and sustain long-term expression of transgenes. The majority of current lentiviral vectors are pseudotyped with the vesicular stomatitis viral envelope (VSV-G). VSV-G equips lentiviral vectors with a broad host cell tropism and increased stability. Increased particle stability enables viral supernatants to be concentrated by high-speed centrifugation to enhance their infectivity. Despite its efficacy, VSV-G is cytotoxic – a feature that prohibits the development of stable cell lines that constitutively express this envelope. Therefore, non-toxic envelope proteins are being investigated. RD114 is an attractive alternative because it also provides increased particle stability and its receptor is widely expressed on hematopoietic stem cells (HSCs). In this study, the packaging efficiency of three envelope proteins, RD114, RDpro and VSV-G, were evaluated with two lentiviral vectors (TRIP GFP and HPV-402). RDpro is an RD114-HIV chimera designed to pseudotype lentiviral vectors more efficiently. In transient systems, VSV-G generated titers of 108 and 107 viral particles/mL for TRIP GFP and HPV-402. RDpro possessed titers of 107 and 106, while RD114 titers were one log lower for each vector. Despite having relatively lower titers, RD114 proteins are less toxic; this was demonstrated in the extension of transient transfection reactions from 48 to 96 h. VSV-G transfections are generally limited to 48 h. In regard to gene therapy applications, we show that RDpro supernatants efficiently transduce peripheral blood HSCs. The versatility of RD114 envelopes was again demonstrated by using a ‘mixed’ expression system; composed of stably expressed RD114 envelope proteins to pseudotype lentiviral vectors generated in trans (titer range 103–105). Our data show that RD114 envelope proteins are effective and versatile constructs that could prove to be essential components of therapeutic lentiviral gene transfer systems.

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