Differentiation of mouse embryonic stem cells after RNA interference-mediated silencing of OCT4 and Nanog.

RNA interference (RNAi) holds great promise as a tool to study the basic biology of stem cells or to direct differentiation in a specific manner. Barriers to achieving efficient and specific gene silencing in RNAi experiments include limitations in transfection efficiency and in the efficacy and specificity of RNAi silencing effectors. Here, we combine methods of efficient lipid-mediated delivery with chemically modified RNAi compounds to silence genes related to pluripotency, in order to direct differentiation of mouse embryonic stem cells. After transfection of embryonic stem cells with OCT4- or Nanog-targeted RNAi compounds, levels of OCT4 or Nanog transcript and protein were reduced accordingly. Reduction in OCT4 expression correlated with induction of trophectoderm genes Cdx2, Hand1, and PL-1, with formation of cells with trophoblast giant cell phenotype after 6 days. Reduction in Nanog expression correlated with induction of extraembryonic endoderm genes GATA4, GATA6, and laminin B1, with subsequent generation of groups of cells with parietal endoderm phenotype. Our results indicate that transient inhibition of OCT4 or Nanog by RNAi compounds is sufficient to induce differentiation toward extraembryonic lineages, which supports the model that these transcription factors function in a dose-dependent manner to influence cell fate.

State-of-the-art modified RNAi compounds for therapeutics.

In recent years, the recognition of non-protein coding RNAs as a functional effector of genetic expression has been highlighted by the discovery of RNA interference (RNAi). RNAi is an intracellular phenomenon that enables the eukaryotic cell to utilize double-stranded RNA molecules to silence gene expression in a sequence-specific manner. The short interfering RNA (siRNA) pathway has been intensively investigated and continues to serve as the basis for the development of potent molecular genetic tools. The power of this technology is most clearly evidenced by the fact that siRNA effector molecules can be chemically synthesized and exogenously delivered to specifically target and silence any gene of choice. This capability enables not only basic research, but also opens the door to a new therapeutic modality. Furthermore, the introduction of certain chemical modifications to siRNA effectors can produce a more robust knockdown of gene expression, hence, optimizing serum stability and increasing target specificity yet limiting the induction of cellular stress response, which are key features for in vivo delivery and successful therapeutics. This article outlines the progress in the development of differentially modified siRNA duplexes and their potential role as human therapeutics.