Inhibition of productive/competitive endocytic pathways enhances siRNA delivery and cell specific targeting

While the use of short interfering RNAs (siRNAs) for laboratory studies is now common practice, development of siRNAs for therapeutic applications has slowed, due in part to a still limited understanding of the endocytosis and intracellular trafficking of siRNA-containing complexes. As a result, it is difficult to design delivery vehicles for specific cell types, resulting in inefficient delivery, cytotoxicity, or immunogenicity when used in vivo. Our aim is to identify which endocytosis and intracellular trafficking pathways lead to active silencing by siRNA-containing complexes. Our work explores the preferential mechanism of endocytosis (whether by clathrin, caveolin, Arf6, Graf1, flotillin, or macropinocytosis) across multiple cell types (HeLa (cervical), H1299 (lung), HEK293 (kidney), and HepG2 (liver)). Using Lipofectamine 2000 (LF2K), fluorescentlylabeled siRNAs were delivered to cells stably expressing green fluorescent protein (GFP). Chemical inhibitors (Filipin, Dynasore, Cytochalasin D, Chlorpromazine, Amiloride, and Methyl-β- cyclodextrin) were used to identify the specific endocytic pathway internalizing the complexes. By measuring the effect of inhibitors on both intracellular levels of siRNA and GFP silencing, we were able to categorize pathways as being productive/competitive according to their functional role in facilitating gene silencing. In productive pathways, siRNAs are actively delivered to a cell and silence a target protein, whereas in competitive pathways, siRNAs are endocytosed but do not lead to silencing. Please click Additional Files below to see the full abstract

Improved asymmetry prediction for short interfering RNA s

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/102096/1/febs12599.pd

Design of siRNA Therapeutics from the Molecular Scale

While protein-based therapeutics is well-established in the market, development of nucleic acid therapeutics has lagged. Short interfering RNAs (siRNAs) represent an exciting new direction for the pharmaceutical industry. These small, chemically synthesized RNAs can knock down the expression of target genes through the use of a native eukaryotic pathway called RNA interference (RNAi). Though siRNAs are routinely used in research studies of eukaryotic biological processes, transitioning the technology to the clinic has proven challenging. Early efforts to design an siRNA therapeutic have demonstrated the difficulties in generating a highly-active siRNA with good specificity and a delivery vehicle that can protect the siRNA as it is transported to a specific tissue. In this review article, we discuss design considerations for siRNA therapeutics, identifying criteria for choosing therapeutic targets, producing highly-active siRNA sequences, and designing an optimized delivery vehicle. Taken together, these design considerations provide logical guidelines for generating novel siRNA therapeutics

Design of siRNA Therapeutics from the Molecular Scale

While protein-based therapeutics is well-established in the market, development of nucleic acid therapeutics has lagged. Short interfering RNAs (siRNAs) represent an exciting new direction for the pharmaceutical industry. These small, chemically synthesized RNAs can knock down the expression of target genes through the use of a native eukaryotic pathway called RNA interference (RNAi). Though siRNAs are routinely used in research studies of eukaryotic biological processes, transitioning the technology to the clinic has proven challenging. Early efforts to design an siRNA therapeutic have demonstrated the difficulties in generating a highly-active siRNA with good specificity and a delivery vehicle that can protect the siRNA as it is transported to a specific tissue. In this review article, we discuss design considerations for siRNA therapeutics, identifying criteria for choosing therapeutic targets, producing highly-active siRNA sequences, and designing an optimized delivery vehicle. Taken together, these design considerations provide logical guidelines for generating novel siRNA therapeutics