Wall Nathan R, Shi Yang
Department of Pathology, Harvard Medical School, Boston, MA 02115, USA.
Lancet. 2003 Oct 25;362(9393):1401-3. doi: 10.1016/S0140-6736(03)14637-5.
RNA interference (RNAi) is the sequence-specific gene-silencing induced by double-stranded RNA (dsRNA), and gives information about gene function quickly, easily, and inexpensively. The use of RNAi for genetic-based therapies is widely studied, especially in viral infections, cancers, and inherited genetic disorders. RNAi has been used to make tissue-specific knockdown mice for studying gene function in a whole animal. Combined with genomics data, RNAi-directed gene-silencing could allow functional determination of any gene expressed in a cell or pathway. The term RNAi came from the discovery that the injection of dsRNAs into Caenorhabditis elegans interferes with the expression of specific genes containing a complementary region to the delivered dsRNA. Although stalled for a time by the non-gene-specific interferon response elicited by dsRNA molecules longer than about 30 nucleotides in mammalian cells, Tom Tuschl's group found that transfection of synthetic 21-nucleotide small-interfering RNA (siRNA) duplexes were highly selective and sequence-specific inhibitors of endogenous genes.
siRNA expression has been studied with siRNA from plasmid and viral vectors that efficiently deliver siRNAs into both dividing and non-dividing cells, stem cells, zygotes, and their differentiated progeny. A collection of RNA interference vectors that suppress 50 human de-ubiquitinating enzymes allowed Thijn Brummelkamp and colleagues to study this gene family and to identify de-ubiquitinating enzymes in cancer-relevant pathways (Nature 2003; 424: 797-801). These researchers found that the familial cylindromatosis tumour suppressor gene (CYLD), previously of unknown function, could enhance the activation of the transcription factor NF-kappaB, leading to increased resistance to apoptosis. They have now started to investigate the use of CYLD inhibitors in clinical trials.
The ability to efficiently and stably produce and deliver sufficient amounts of siRNA to the proper target tissues require refinement before this new technology can be tried clinically. Initial in-vivo studies reported effective transgene suppression in adult mice by chemically synthesised siRNAs. More recently many researchers have used plasmid and viral vectors for transcription of short-hairpin RNAs, both in vitro and in vivo. With these expression systems, gene expression was more stably inhibited than with the transient knockdown recorded with chemically synthesised siRNA. Human trials exploiting these latest findings are likely to soon follow.
RNA干扰（RNAi）是由双链RNA（dsRNA）诱导的序列特异性基因沉默，能快速、简便且低成本地提供有关基因功能的信息。RNAi在基于基因的治疗中的应用得到了广泛研究，尤其是在病毒感染、癌症和遗传性疾病方面。RNAi已被用于制作组织特异性基因敲除小鼠，以在整体动物中研究基因功能。结合基因组学数据，RNAi介导的基因沉默可以确定细胞或通路中任何表达基因的功能。RNAi这个术语源于这样一个发现：将dsRNA注射到秀丽隐杆线虫中会干扰含有与导入的dsRNA互补区域的特定基因的表达。尽管在哺乳动物细胞中，长度超过约30个核苷酸的dsRNA分子引发的非基因特异性干扰素反应曾一度阻碍了相关研究，但汤姆·图斯克的团队发现，转染合成的21个核苷酸的小干扰RNA（siRNA）双链体是内源性基因的高度选择性和序列特异性抑制剂。
人们已利用来自质粒和病毒载体的siRNA对siRNA表达进行了研究，这些载体能有效地将siRNA导入分裂细胞和非分裂细胞、干细胞、受精卵及其分化后代。蒂恩·布鲁梅尔坎普及其同事利用一组抑制50种人类去泛素化酶的RNA干扰载体，对这个基因家族进行了研究，并在与癌症相关的通路中鉴定出去泛素化酶（《自然》，2003年；424：797 - 801）。这些研究人员发现，此前功能未知的家族性圆柱瘤肿瘤抑制基因（CYLD）可以增强转录因子NF-κB的激活，从而增加对细胞凋亡的抗性。他们现在已开始在临床试验中研究CYLD抑制剂的应用。
在这项新技术能够进行临床尝试之前，需要改进有效且稳定地产生并向适当靶组织递送足够量siRNA的能力。最初的体内研究报告称，化学合成的siRNA可有效抑制成年小鼠体内的转基因表达。最近，许多研究人员已在体外和体内使用质粒和病毒载体转录短发夹RNA。使用这些表达系统时，基因表达的抑制比化学合成的siRNA所记录的瞬时敲除更稳定。利用这些最新发现的人体试验可能很快就会开展。
