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Tools & Techniques: Lipids for siRNA Delivery to the Lung; Software for Small RNA Analysis; and More


Amid a strong interest in developing new delivery technologies for RNAi drugs, a research team from Oregon State University has developed non-structured lipids capable of carrying a combination of siRNAs and a chemotherapeutic agent directly into the lung.

Chemotherapy remains a key part of lung cancer treatment, but systemic treatments often cause adverse effects in healthy organs, while orally delivered therapies are limited by poor bioavailability in the lung. Meanwhile, although some existing chemotherapies have been formulated for inhalation, they are limited in number and still suffer from tumor resistance.

To address these issues, the investigators developed nanostructured lipid carriers capable of delivering the chemotherapeutic compounds doxorubicin and paclitaxel, as well as siRNAs designed to inhibit two proteins associated with tumor cell resistance — MRP1 and BCL2.

When tested in animal models of lung cancer, inhalation of the carriers led to their preferential accumulation in the lungs compared with accumulation in other organs, the scientists wrote in a paper published in the Journal of Controlled Release. In comparison, intravenous injection of the same nanosystem led to predominant accumulation in the liver, kidney, and spleen, with significantly less accumulation in the lungs.

“Such passive targeting by the local delivery to the lung substantially limited possible adverse side effects on healthy organs,” they wrote.

To further target the molecules specifically to lung cancer cells, the team then incorporated a synthetic analog of LHRH peptide as a targeting moiety, having previously found that LHRH receptors are overexpressed in many cancer cells including lung cancer cells, but not in healthy visceral organs.

They found that inclusion of the peptide boosted drug accumulation in lung cancer cells following inhalation, supporting the combination of passive lung targeting via inhalation and active targeting using the LHRH peptide, according to the paper.

Additional analyses confirmed that the delivery system carried its payload into cancer cells, and that treatment induced cancer cell death while slowing or halting tumor progression in the animal models.

“Therefore, the proposed [nanosystem] effectively performed its multifunctional task providing simultaneous cell death induction and suppression of drug resistance in lung cancer cells,” the researchers concluded in the paper.

To help deal with the large amounts of data that are produced from the deep sequencing of viral small RNAs, a group of UK researchers has developed a software package to condense, analyze, and display such data.

Software packages for analyzing viral small RNAs exist, but can only be used on certain operating systems and often require advanced programming skills, the researchers wrote in Bioinformatics.

To overcome these issues, the team developed viRome, a package for R that allows for the “interactive generation of a range of informative plots and reports,” according to the paper.

Among its advantages over existing tools, the software allows interaction between the user and the software during report and graph generation; is available on any operating system that supports R including Microsoft Windows and Linux; and separates visualization from alignment, which allows the use of any alignment software, the paper adds.

While existing shRNA repositories offer access to constructs suitable for genome-wide and more focused loss-of-function screens, sometimes the RNAi molecules are not sufficiently effective and reliable.

To provide an alternative source of shRNAs for such cases, a team led by Brandeis University investigators has developed a do-it-yourself approach for the rational design of custom oligos called the organic shRNA platform, or OshR.

“The OshR platform is ‘organic’ because it conforms more naturally to the endogenous vertebrate miRNAs by maintaining specific bulges and incorporating strategic mismatches to insure the desired guide strand is produced while reducing the accumulation of passenger strands that might contribute to off-target effects,” the scientists wrote in Methods.

According to the paper, the OshRNAs are effective in specifying the production of the desired guide strand because they incorporate natural miRNA-30 features to “consistently suppress passenger strand accumulation.” In cell culture experiments, the researchers demonstrated that the platform’s reliability is further increased when sequences target the 3’ UTRs of a gene.

Overall, the platform offers an attractive option for researchers looking for ways to construct a small pool of custom shRNAs, the team concluded.

Aiming to improve researchers’ ability to integrate microRNA and mRNA expression data, a multi-institute team of German investigators has developed a new algorithm for identifying miRNA/mRNA regulation between tumor tissue samples.

Called Classification-based Analysis of Paired Expression Data of RNA, or CAPE RNA, the algorithm can assign interaction states to each sample without preexisting stratification of groups, the scientists wrote in PLoS One. “The distribution of the assigned interaction states compared to given experimental groups is used to assess the quality of a predicted interaction.”

As a proof of concept, the team used CAPE RNA to analyze samples of bladder cancer and normal bladder tissue derived from 24 patients.

Normal and cancer tissues, as well as different stages of tumor progression, were successfully stratified, and data from the study point to potential miRNA/mRNA interactions that may be associated with tumor progression.

Given the need for ways to verify target protein knockdown in RNAi experiments, investigators from the University of Washington have developed a targeted proteomics approach that can assess such protein depletion in human cell and Drosophila.

The method is based on selected reaction monitoring, or SRM, mass spectrometry and detects low-abundance peptides that can be used as a proxy for a target protein, according to a report in the Journal of Proteome Research.

To demonstrate the approach, the team used it to detect the effect of RNAi on proteins that “represent analytical challenges,” they wrote. These include the human tumor suppressor protein TP53, which is “variably expressed and is modified across a wide range of residues by post-translational modifications,” and WRN, a low-abundance, DNA-binding protein that is lost in Wener syndrome.

They also looked at two Drosophila proteins that are short accessory gland seminal fluid proteins among the more than 130 seminal fluid proteins transferred from males to females during mating.

The researchers were able to develop an SRM protocol that could detect and identify differences in the abundance of the four proteins following RNAi in human cells and whole flies, with “excellent agreement” between the method and Western blot-based detection and quantitation methods, according to the paper.