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

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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.