治療性的慢病毒載體已變成分子醫(yī)學(xué)上重要的工具,,越來(lái)越多的臨床試驗(yàn)都使用這種載體系統(tǒng),,從基礎(chǔ)研究的觀點(diǎn)來(lái)看,,慢病毒載體是令人著迷的物質(zhì),,一方面,,第一型人類免疫缺陷病毒(HIV)可以提供較大的容量及能力將基因轉(zhuǎn)殖到不分裂的細(xì)胞如骨髓干細(xì)胞(HSCs) 及神經(jīng)細(xì)胞,。但一些顧慮如可能產(chǎn)生會(huì)復(fù)制的病毒顆粒,,使得我們必須移除HIV基因的一大部份,主要是外套膜蛋白質(zhì),。在十月份《實(shí)驗(yàn)生物及醫(yī)學(xué)》(Experimental Biology and Medicine) 期刊發(fā)表的特色文章,,Bell等人發(fā)現(xiàn)來(lái)自內(nèi)生性貓病毒RD114的外套膜蛋白質(zhì)可以提供一個(gè)有效及多樣的方法偽態(tài)化慢病毒載體,這工作由Anthony Bell執(zhí)行,,和哥倫比亞大學(xué)醫(yī)學(xué)院的David Fegen,、Maureen Ward、及Arthur Bank共同合作,。
Bell博士說(shuō)明" 大部份的慢病毒載體都是偽態(tài)的或包裹在水泡性口炎病毒(VSV-G) 的外套膜內(nèi),,VSV-G讓慢病毒載體可以有較廣的細(xì)胞感染性,也較穩(wěn)定,,病毒顆粒較高的穩(wěn)定性可以讓病毒液經(jīng)高速離心濃縮以增加感染性,,但VSV-G對(duì)細(xì)胞有毒性,使得帶此外套膜的偽態(tài)慢病毒載體無(wú)法持續(xù)的表達(dá),。因此我們研究不具毒性的病毒外套膜如RD114,,RD114是有特別吸引力的另一種選擇,,因?yàn)檫@個(gè)外套膜也同樣有高穩(wěn)定性,HSCs也表達(dá)受體可以被感染,。因此RD114可以被證明是治療慢病毒載體系統(tǒng)的一個(gè)重要成員,。
這個(gè)研究團(tuán)隊(duì)比較三個(gè)外套膜和二個(gè)慢病毒載體的包裝效率:VSV-G、RD114,、及RDpro,,RDpro是一個(gè)RD114-HIV嵌合體設(shè)計(jì)為高效率的偽態(tài)慢病毒載體,在短暫的表達(dá)下,,如預(yù)期的VSV-G有最高的力價(jià),,但非毒性的RD114能使得一般的轉(zhuǎn)感染反應(yīng)時(shí)間從48小時(shí)延長(zhǎng)到96小時(shí),VSV-G的轉(zhuǎn)感染通常限制在48小時(shí),,因此RD114外套膜比起VSV-G提供一個(gè)有效的方法可以增加慢病毒產(chǎn)出的總量,。RD114的多樣性也經(jīng)更進(jìn)一步的探討表現(xiàn)出來(lái),利用穩(wěn)定產(chǎn)出的RD114蛋白質(zhì),,偽態(tài)慢病毒載體再經(jīng)由反式的作用,,產(chǎn)生" 混合的"慢病毒表達(dá)系統(tǒng)。
Bell博士說(shuō)明"我們對(duì)于混合表達(dá)系統(tǒng)的方式所得到的結(jié)果很興奮,,我們也很驚訝地發(fā)現(xiàn)RD114蛋白質(zhì)的表現(xiàn)比嵌合體的RDpro好,。在基因治療方面,RDpro懸浮液可以有效的轉(zhuǎn)染周邊血液HSCs,,我們相信這程度的轉(zhuǎn)染可以再經(jīng)濃縮步驟優(yōu)化后而增強(qiáng),。"
《實(shí)驗(yàn)生物及醫(yī)學(xué)》期刊主編Steven R. Goodman說(shuō) "Anthony Bell及其同事令人興奮的研究證明了使用不具毒性的外套膜蛋白質(zhì)是偽態(tài)慢病毒載體的另一種選擇,它對(duì)未來(lái)的基因治療方式有很大的潛力,。"(生物谷Bioon.com)
生物谷推薦英文摘要:
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.