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Therapeutic agents acting on RNA, including RNA modification, RNA editing and RNA interference (RNAi), play a vital role in gene function study, signaling pathway, drug discovery, disease treatment, vaccine development and so on. Therein, RNAi as an emerging gene therapy has been widely applied in many cancer studies by silencing oncogenes or specific mRNA in malignant tumor cells. Mechanism and efficiency of RNAi are the key issues of RNAi technology. RNA silencing involves dynamic modeling, analyses and optimal control of RNAi gene system. Physiological delay and Hill response describing off-target are considerable elements involving RNAi efficiency. In this paper, we first formulate a four-dimensional RNAi model with time delays and Hill functions, and then investigate the complex dynamic behaviors including the number, existence and stability of internal equilibria and Hopf bifurcations of single delay and two delays. Furthermore, based on the specific mRNA degradation adopted in impulsive patterns, we build an optimal problem by adding exogenous dsRNA at alterable time points in variable dosages in a treatment session. By the method of gradient formula, we can find the optimal impulsive time and proportion of dsRNA. Finally, simulation indicates that (1) physiological lags not only raise the oscillations of mRNA but also cut down the levels of cost; (2) smaller delays and larger rates of siRNA–mRNA complex formation and dsRNA synthesis imply the rapid composition of RISC and fast synthesis of dsRNA leading to more desirable therapeutic schedule, which affords evidence for gene regulation and RNAi; (3) a larger half-saturation coefficient characterizes a unique and stable higher targeted mRNA, whereas a smaller half-saturation coefficient generates bistability in which the higher and lower targeted mRNAs simultaneously emerge; and (4) the bistability will provide a good guidance to control, suppress and degrade targeted mRNA.

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2006 Nobel Prize in Physiology/Medicine — Professor Craig C. Mello and Professor Andrew Z. Fire.

Design of small interference RNA (siRNA) is one of the most important steps in effectively applying the RNA interference (RNAi) technology. The current siRNA design often produces inconsistent design results, which often fail to reliably select siRNA with clear silencing effects. We propose that when designing siRNA, one should consider mRNA global features and near siRNA-binding site local features. By a linear regression study, we discovered strong correlations between inhibitory efficacy and both mRNA global features and neighboring local features. This paper shows that, on average, less GC content, fewer stem secondary structures, and more loop secondary structures of mRNA at both global and local flanking regions of the siRNA binding sites lead to stronger inhibitory efficacy. Thus, the use of mRNA global features and near siRNA-binding site local features are essential to successful gene silencing and hence, a better siRNA design. We use a random forest model to predict siRNA efficacy using siRNA features, mRNA features, and near siRNA binding site features. Our prediction method achieved a correlation coefficient of 0.7 in 10-fold cross validation in contrast to 0.63 when using siRNA features only. Our study demonstrates that considering mRNA and near siRNA binding site features helps improve siRNA design accuracy. The findings may also be helpful in understanding binding efficacy between microRNA and mRNA.

The development of nanoscale delivery vehicles for siRNAs is a current topic of considerable importance. However, little is understood about the exact trafficking mechanisms for siRNA-vehicle complexes across the plasma membrane and into the cytoplasm. While some information can be gleaned from studies on delivery of plasmid DNA, the different delivery requirements for these two vehicles makes drawing specific conclusions a challenge. However, using chemical inhibitors of different endocytosis pathways, studies on which endocytotic pathways are advantageous and deleterious for the delivery of nucleic acid drugs are emerging. Using this information as a guide, it is expected that the future development of effective siRNA delivery vehicles and therapeutics will be greatly improved.

The regulation of embryonic stem (ES) cell self-renewal and pluripotency is based upon highly orchestrated transcription factor networks. RNA inhibition has been demonstrated to affect ES cell function by altering gene expression levels that are critical to the maintenance and differentiation of ES cells. Fragile X mental retardation protein (FMRP) is a selective RNA-binding protein that can act as a translational repressor for bound mRNA and regulates the expression of a variety of gene transcripts in numerous adult cells. The absence of FMRP results in the most common form of inherited intellectual disability, Fragile X syndrome. In an effort to determine the role of FMRP during development, we silenced the FMRP gene (FMR1) using short hairpin RNA (shRNA). Prior to differentiation induction, we analyzed the phenotype of FMR1 knock down (FMR1-kd) mouse ES cells in their undifferentiated state. Herein, we report that FMR1-kd ES cells proliferate at a greater rate than wild-type ES cells resulting in a 25% reduction in doubling time. FMR1-kd ES cells were found to have an increased expression of three self-renewal genes (OCT-4, Sox2, Nanog) in the undifferentiated state. Moreover, FMR1-kd ES cells failed to downregulate OCT-4 during differentiation programs resulting in abnormal fate decisions in vitro. These results demonstrate an unexpected correlation between FMR1 expression and OCT-4 regulation suggesting that FMRP is involved in the silencing of OCT-4 during the commencement of differentiation programs.

Understanding the endocytosis and intracellular trafficking of short interfering RNA (siRNA) delivery vehicle complexes remains a critical bottleneck in designing siRNA delivery vehicles for highly active RNA interference (RNAi)-based therapeutics. In this study, we show that dextran functionalization of silica nanoparticles enhanced uptake and intracellular delivery of siRNAs in cultured cells. Using pharmacological inhibitors for endocytotic pathways, we determined that our complexes are endocytosed via a previously unreported mechanism for siRNA delivery in which dextran initiates scavenger receptor-mediated endocytosis through a clathrin/caveolin-independent process. Our findings suggest that siRNA delivery efficiency could be enhanced by incorporating dextran into existing delivery platforms to activate scavenger receptor activity across a variety of target cell types.