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Tools & Techniques: siRNA Delivery and Modification, Synthetic microRNA Circuits


NEW YORK (GenomeWeb) – As part of an effort to advance conjugate-based drug-delivery technologies for siRNA therapeutics, researchers from China's Southern Medical University this month reported in vivo data on a new approach for tumor-targeted gene silencing.

The paper, which was co-authored with collaborators at RiboBio and appeared in Nucleic Acids Research, point to the potential of conjugate approaches for RNAi in areas outside of the liver, where the most of the work with such delivery technologies have been focused.

The study focused on the cell surface receptor integrin alpha-v beta-3, which has been shown to play a role in angiogenesis and is upregulated in tumor blood vessels in a number of cancers. Given its high affinity of the receptor, arginine–glycine–aspartate (RGD) peptide has been examined by a variety of groups as a ligand for tumor-targeting liposomes.

Theorizing that inhibition of a tumor's ability to create new blood vessels could slow its growth, the scientists constructed siRNAs targeting vascular endothelial growth factor receptor 2, which were tested in vitro for their ability to silence their intended target.

The siRNAs were then linked to the RGD peptides and shown to be able to enter cells expressing the integrin alpha-v beta-3 receptor without the need for a transfection reagent, triggering an RNAi effect. Microinjection of zebrafish blastocysts with the agent, meantime, resulted in specific inhibition of blood vessel growth

The scientists then tested their compound in tumor-bearing mice, showing that the conjugates distributed to tumors after systemic administration without triggering an immune response and led to a specific downregulation of their target gene.

Moreover, treatment led to a reduction in blood vessel formatting in the mice, as well as substantially slower tumor growth, providing proof of concept for developing RGD peptides as carriers of therapeutic siRNAs for solid tumors, the researchers wrote in their report.

They cautioned, however, that additional studies are still needed before taking the delivery approach toward the clinic.

Aiming to improve upon the potency and deliverability of conventional siRNAs, a group of Korean researchers has developed a new RNAi agent dubbed a tripodal interfering RNA (tiRNA), and this month published data demonstrating the use of a novel liver-targeting delivery system for the molecules.

Notably, tiRNAs, which are formed by the complementary association of three single-stranded RNAs, have been shown capable of silencing multiple target genes in a Dicer-independent manner. The latest data, which appeared in the Journal of Controlled Release suggest that these molecules may be effective in treating liver-specific diseases.

In their study, the investigators generated tiRNAs targeting three different genes, including apolipoprotein B (apoB), with the 5' region of the molecules' antisense strands oriented toward the outside, which enables their multi-target knockdown capabilities. These were then combined with linear, high-molecular weight polyethyleneimine (PEI) that was functionalized with galactose in order to facilitate its liver uptake via asialoglycoprotein receptors.

In mice, the tiRNA molecules were found to localize in the liver and induce apoB by as much as 63 percent after a single dose compared with a 33 percent reduction with apoB-targeting siRNAs delivered using the same PEI/galactose complex. No silencing effect was observed in control animals. Further, serum cholesterol levels were reduced in animals receiving the tiRNA treatment.

A team of Japanese scientists has reported this month on the use of an artificial nucleic acid to boost an siRNAs stability while suppressing sense strand loading into RISC.

Called a serinol nucleic acid (SNA), the acyclic nucleic acid is created through the replacement of a ribose backbone with a 2-aminopropane-1, 3-diol scaffold. Although its chemical structure is different from natural DNA or DNA, SNAs pair sequence-specifically with both, according to a paper appearing in ChemBioChem. "Furthermore, SNA forms a more stable hybrid with RNA than with DNA."

In their report, the investigators introduced SNAs at the terminal positions of both termini of siRNAs' sense strands, as well as at the 3' terminus of the antisense strand. Doing so, they reported, significantly increased strand selectivity in RISC formation and resistance to enzymatic digestion.

Given that they tested their SNA-modified siRNAs against reporter genes, the team wrote that experiments with endogenous genes are warranted.

Looking to fill a gap in microRNA research, a team from Stanford University has developed a quantitative framework for the forward engineering of synthetic miRNA circuits.

According to a paper appearing in Nature Methods, the model captures the quantitative relationship between miRNA and target gene expression levels as a function of parameters, including mRNA half-life and miRNA target-site number.

This model was extended to predict the behavior of synthetic circuits that incorporate protein-responsive miRNA switches, and the scientists designed an "optimized miRNA-based protein concentration detector circuit that noninvasively measures small changes in the nuclear concentration of beta-catenin owing to induction of the Wnt signaling pathway," they wrote.