An innovative strategy for the efficient cytosolic delivery of small interfering RNA (siRNA)

RNA干扰在本世纪初被概念化并得到证实，并于2006年获得诺贝尔生理学和医学奖。此后，利用RNA干扰进行治疗的热潮不断涌现。RNA干扰是一种天然存在的高度保守的生物机制，它通过降解一类重要的细胞分子来调节细胞功能，这些分子被称为信使核糖核酸(Messenger Ribonna，mRNA)。信使核糖核酸的降解发生在胞浆内的细胞内，胞浆是一个流动的细胞室。在细胞质中，mRNA的降解是通过与小干扰RNA(SiRNA)的相互作用来调节的，小干扰RNA是自然产生的分子。它们能够使大量不同的mRNA失活，从而使RNA干扰对各种疾病具有巨大的治疗潜力，如高胆固醇血症、病毒感染或癌症。对于治疗应用，人工合成的siRNA被整合到靶向纳米尺寸的运输载体中，这有助于运输载体与靶细胞的特定结合。在内吞进入细胞后，载体被输送到内体运输系统，这是一个复杂的、孤立的管状-囊泡室网络。这些管状囊泡间隔被胞浆包围。为了启动RNA干扰，siRNA必须从这些隔间逃逸到细胞质中，这一过程被称为内体逃逸。SiRNA的内体逃逸不足是RNA干扰治疗成功的制约因素之一，阻碍了基于siRNA的药物的整体疗效。因此，研究能够促进siRNA内体逃逸的特定细胞内递送策略将是siRNA递送领域的一大进步。在计划的项目中，将研究植物来源的递送系统(称为协同原理)的潜力，以将siRNA定向递送到细胞质中。作为一种进化优化的过程，协同原理是文献中描述的最有效的传递调制系统之一。这一系统的基础是某些植物次生代谢物(如皂苷SA1641)以高度特异性的方式特异性地触发特定类别的植物衍生酶(Saporin)的内体逃逸。因此，在拟议的项目中，设想产生掺入SA1641的纳米载体中的siRNA，并研究SA1641介导的siRNA的内体逃逸。另一种实验策略是产生一种酶失活的Saporin变体(SapKQ)作为siRNA的拖拽分子，并将SapKQ-siRNA结合物和SA1641整合到靶向纳米载体中，然后分析SA1641介导的SapKQ-siRNA结合物内体逃逸到细胞质中的情况。

RNA-interference was conceptualized and proven in the beginning of this century and was honored by the Nobel Prize for physiology and medicine in 2006. Thereafter, there has been tremendous upsurge to utilize RNA-interference for therapeutic applications. RNA-interference is a naturally occurring highly conserved biological mechanism that modulates cellular functions by the degradation of an important class of cellular molecules, which are known as messenger ribonucleic acid (mRNA). The degradation of mRNA takes place in the cell within the cytosol, which is a fluidic cellular compartment. In the cytosol the degradation of mRNA is mediated by the interaction with small interfering RNAs (siRNA), which are naturally occurring molecules. Their ability to inactivate a huge number of different mRNA and thus cellular functions makes RNA-interference of great therapeutic potential for diverse diseases, such as hypercholesterolemia, viral infections or cancer. For therapeutic applications, synthetic siRNA is integrated into a targeted nano-sized transport vehicle, which facilitates the specific binding of the transport vehicle to the target cell. After endocytosis into the cells the vehicle is delivered into the endosomal transport system, which is a complex, isolated network of tubular-vesicular compartments. These tubular-vesicular compartments are surrounded by the cytosol. To initiate RNA-interference siRNA has to escape from these compartments into the cytosol, a process, which is designated as endosomal escape. The insufficient endosomal escape of siRNA is one of the restricting factors for the therapeutic success of RNA-interference and hampers the overall efficacy of siRNA based drugs. The investigation of specific intracellular delivery strategies that could facilitate the endosomal escape of siRNA would therefore be a great step forward in the field of siRNA delivery. In the intended project the potential of a plant derived delivery system (termed as synergistic principle) will be investigated for the targeted delivery of siRNA into the cytosol. An evolutionarily optimized process, the synergistic principle is one of the most efficient delivery modulating systems ever described in literature. The basis for this system is the ability of certain plant secondary metabolites (e.g. Saponin SA1641) to specifically trigger the endosomal escape of a particular class of plant-derived enzymes (Saporin) in a highly specific manner. Therefore in the proposed project it is envisaged to generate siRNA incorporated in nano-vehicles with SA1641 and to investigate the SA1641-mediated endosomal escape of siRNA. Another experimental strategy is the generation of an enzymatically inactive variant of Saporin (SapKQ) as a drag molecule for siRNA and the integration of SapKQ-siRNA conjugates and SA1641 into targeted nano-vehicles followed by the analysis of the SA1641-mediated endosomal escape of SapKQ-siRNA conjugates into the cytosol.