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.

While small interfering RNAs (siRNAs) are commonly used for laboratory studies, development of siRNA therapeutics has been slower than expected, due, in part, to a still limited understanding of the endocytosis and intracellular trafficking of siRNA-containing complexes. With the recent characterization of multiple clathrin-/ caveolin-independent endocytic pathways, that is, those mediated by Graf1, Arf6, and flotillin, it has become clear that the endocytic mechanism influences subsequent intracellular processing of the internalized cargo. To explore siRNA delivery in light of these findings, we developed a novel assay that differentiates uptake by each of the endocytic pathways and can be used to determine whether endocytosis by a pathway leads to the initiation of RNA interference (RNAi). Using Lipofectamine 2000 (LF2K), we determined the endocytosis pathway leading to active silencing (whether by clathrin, caveolin, Arf6, Graf1, flotillin, or macropinocytosis) across multiple cell types (HeLa, H1299, HEK293, and HepG2). We showed that LF2K is internalized by Graf1-, Arf6-, or flotillin-mediated endocytosis for the initiation of RNAi, depending on cell type. In addition, we found that a portion of siRNA-containing complexes is internalized by pathways that do not lead to initiation of silencing. Inhibition of these pathways enhanced intracellular levels of siRNAs with concomitant enhancement of silencing.

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.

In the development of RNA interference (RNAi) therapeutics, merely selecting short, interfering RNA (siRNA) sequences that are complementary to the messenger RNA (mRNA) target does not guarantee target silencing. Current algorithms for selecting siRNAs rely on many parameters, one of which is asymmetry, often predicted through calculation of the relative thermodynamic stability of the two ends of the siRNA. However, we have previously shown that highly-active siRNA sequences are likely to have particular nucleotides at each 5’-end, independent of their thermodynamic asymmetry. Here, we describe an algorithm for predicting highly active siRNA sequences based only on these two asymmetry parameters. The algorithm uses end sequence nucleotide preferences and predicted thermodynamic stabilities, each weighted based on training data from the literature, to rank the probability that an siRNA sequence will have high or low activity. The algorithm successfully predicts weakly- and highly-active sequences for enhanced green fluorescent protein (EGFP) and protein kinase R (PKR). Use of these two parameters in combination improves the prediction of siRNA activity over current approaches for predicting asymmetry. Going forward, we anticipate that this approach to siRNA asymmetry prediction will be incorporated into the next generation of siRNA selection algorithms.

Under physiological conditions, phosphatidylserine (PS) predominantly localizes to the cytosolic leaflet of the plasma membrane of cells. During apoptosis, PS is exposed on the cell surface and serves as an “eat-me” signal for macrophages to prevent releasing self-immunogenic cellular components from dying cells which could potentially lead to autoimmunity. However, increasing evidence indicates that viable cells can also expose PS on their surface. Interestingly, tumor cell-derived extracellular vesicles (EVs) externalize PS. Recent studies have proposed PS-exposing EVs as a potential biomarker for the early detection of cancer and other diseases. However, there are confounding results regarding subtypes of PS-positive EVs, and knowledge of PS exposure on the EV surface requires further elucidation. In this study, we enriched small EVs (sEVs) and medium/large EVs (m/lEVs) from conditioned media of breast cancer cells (MDA-MB-231, MDA-MB-468) and non-cancerous cells (keratinocytes, fibroblasts). Since several PS-binding molecules are available to date, we compared recombinant proteins of annexin A5 and the carboxylated glutamic acid domain of Protein S (GlaS), also specific for PS, to detect PS-exposing EVs. Firstly, PS externalization in each EV fraction was analyzed using a bead-based EV assay, which combines EV capture using microbeads and analysis of PS-exposing EVs by flow cytometry. The bulk EV assay showed higher PS externalization in m/lEVs derived from MDA-MB-468 cells but not from MDA-MB-231 cells, while higher binding of GlaS was also observed in m/lEVs from fibroblasts. Second, using single EV flow cytometry, PS externalization was also analyzed on individual sEVs and m/lEVs. Significantly higher PS externalization was detected in m/lEVs (annexin A1+) derived from cancer cells compared to m/lEVs (annexin A1+) from non-cancerous cells. These results emphasize the significance of PS-exposing m/lEVs (annexin A1+) as an undervalued EV subtype for early cancer detection and provide a better understanding of PS externalization in disease-associated EV subtypes.