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【原创】外源性siRNA的合成和体外导入方法4

丁香园论坛

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第四节 病毒载体携带的siRNA
介绍
在大多数真核细胞,RNAi是一个高度保守的机制,它被认为是基因表达和抗病毒机制的调节剂。1-4 RNAi是一个多级的过程,第一步是:在一种核糖核酸酶Ⅲ样的酶(Dicer5 )的作用下,双链RNA(dsRNA)被裂解成小干扰RNA(siRNA)。6这些21到23个核苷酸的siRNA随后参与构成RNA诱导的沉默复合物(RISC),可以将siRNA带到同源mRNA的地方,并将其破坏。
在2001年,Elbashir 等 发现用化学合成的长21个核苷酸的siRNA瞬时转染哺乳动物细胞,可以获得基因特异性抑制。这些siRNA的长度足以诱导产生基因特异性抑制,又不至于引起宿主的干扰反应。另外,siRNA也可以来自于含有茎环发夹结构的 dsRNA。这些短发夹RNA(shRNAS) 可以用含有RNA聚合酶Ⅲ(PolⅢ)启动子的质粒DNA在体外表达。9-14 接着shRNA被Dicer切割成siRNA,然后在胞浆参与构成RISC,降解mRNA。这些研究表明在哺乳动物细胞产生 si/shRNA是可行的。然而,沉默效果是短暂的,转染原代细胞效率相对较低。为了提高原代细胞的转导效率,使siRNA长期稳定表达,人们已经改造病毒载体作为导入siRNA的工具。

导入shRNA的病毒载体
长期的基因抑制对于决定基因的功能是必要的,需要一种有效地方法建立一系列的细胞系,转基因动物,和使靶基因稳定沉默的治疗策略。目前一些组织已经利用病毒载体系统在多种细胞和动物成功地介导了长期的基因特异性抑制, 这样,将RNAi 作为一种治疗手段用于动物模型就更可行了。下面我们将列出为siRNA基因治疗设计的不同病毒载体的优缺点。
表达shRNA的腺病毒载体已经在体内和体外成功地降低了靶蛋白的水平。15,16 这个载体系统提高了将 shRNA导入多种组织细胞的效率。17 不过,由于腺病毒载体在细胞内保持游离,并且随着细胞的分裂而稀释,所以shRNA的表达是短暂的。腺病毒蛋白的低水平表达,以及对腺病毒蛋白的预存免疫也导致了表达shRNA的细胞数目的减少,限制了它在体内的应用。
腺相关病毒(AAV)载体系统很有应用前景,既能转染分裂细胞,又能转染非分裂细胞,而且不会致病,不会激发免疫反应。它可以以游离的形式在转染细胞长期存在,偶尔可以整合到宿主基因组里,使导入的基因长期表达。尽管AAV载体能高效转导肌细胞和脑细胞,但是用来转导造血干细胞却很差,而且存在不少争议,限制了AAV载体的应用。
既然致癌逆转录病毒载体(Oncoretroviral vector)可以整合到宿主细胞,建立稳定的基因转导,人们已经将其由于许多实验室研究和人类基因治疗的临床试验。21 目前有许多机构可以提供表达shRNA的致癌逆转录病毒载体(Oncoretroviral vector)。9,22,24 致癌逆转录病毒载体的主要缺点是不能转导非分裂细胞,这就使它的应用范围大大缩小。25 而且,经常观察到转基因表达的转录失活现象。26 在治疗应用上,致癌逆转录病毒载体有一个很严重的缺点,就是它有可能通过插入突变激活细胞的癌基因,导致恶性肿瘤。
在过去的几年里,慢病毒载体介导的基因转移已经成功地用于多种类型的细胞,包括原代细胞和组织。27 因为慢病毒载体有核定位信号,所以它可以转导分裂细胞。28,29 目前已经从多种逆转录病毒构建了逆转录病毒载体,一些研究者把目光投向了以Ⅰ型为人免疫缺损病毒(HIV-1)为代表的慢病毒。30 由于加入了HIV-1中央聚嘌呤区(cppt)元件,提高了载体整合前核转位的效率。31 土拨鼠肝炎病毒转录后调节元件(WPRE)也会增加转录后转基因的表达。32 为了确保载体的安全性,HIV-1复制所必需的gag/pol 和rev基因由两个不同的质粒DNA表达。33 去除自身灭活载体LTRs中的病毒增强子和启动子,可以使慢病毒载体的安全性进一步提高。33 为了使其可以转导更多的细胞类型,载体中可以加入不同的包膜和细胞表面分子。其中, 疱疹性口炎蛋白G(VSV-G)有最广泛的宿主群。 34 将逆转录病毒载体直接注射到啮齿类动物体内,在多种器官可以有持久的转基因表达,包括脑,肌肉,肾,和视网膜。29,35 在多种动物模型,逆转录病毒载体能够在没有细胞因子的刺激下转导造血干细胞或造血祖细胞,使转入的基因在造血细胞的多重分化谱系中更有效地表达。36-38慢病毒也可以转染巨噬细胞和树突细胞38,39与传统的原核注射/同源重组技术相比,利用慢病毒载体的转基因鼠的生产要有效得多。40,41与致癌逆转录病毒载体不同,慢病毒载体对转基因失活高度抵抗,在转基因鼠可以持续稳定的表达。40因此,慢病毒载体在基因治疗中应该有很广的应用范围。 我们与合作者已经开发了递呈si/shRNA的慢病毒载体,并且成功地使目的基因沉默,包括CCR5化学因子受体基因和HIV-1调节因子基因38,42-51它还可以靶向恶性肿瘤进展过程中的一个关键的基因,p53,这也证明了它作为抗肿瘤药物的巨大潜力。研究表明,以HIV-1为基础构建的这类慢病毒载体具有可感染非分裂细胞、目的基因整合至靶细胞基因组长期表达、免疫反应小等优点,适于体内基因治疗,因此有望成为理想的基因转移载体。

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