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Tools & Techniques: Screening siRNA Delivery Nanoparticles; Reporter Model for siRNA PD; and More

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In an effort to improve the screening and selection of lipid nanoparticle delivery vehicles for siRNA therapeutics, researchers from Merck have developed an in vitro assay designed to test how the particles will behave in vivo.

After systemic administration, lipid nanoparticles are exposed to a variety of biological elements such as serum proteins and blood proteins that can affect their ability to effectively carry RNAi payloads to target tissues.

Given the cost of testing delivery particles in animals, various in vitro assays have been developed to screen lipid nanoparticles for their serum stability, endosomal escape propensity, and other critical features. Yet none have proven particularly useful primarily because "the in vitro environment fails to adequately approximate the in vivo situation," the Merck team wrote in the Journal of Controlled Release.

To address this, the Merck team developed a single assay that incorporates all of the major environmental changes that lipid nanoparticles encounter after systemic administration, specifically focusing on serum interactions, acidic endosomal pH, and anionic endosomal lipids.

Nanoparticles are incubated with 50 percent serum from different species at 37 degrees Celsius, they wrote in their paper. The resulting samples were then incubated with anionic liposomes, approximating the endosomal membrane, at pH 6 and 7.5.

"The amount of siRNA freed from the [nanoparticles] after these treatments serves as a measure for the effectiveness of delivery," they added.

Experiments with the assay indicated that the amount of siRNA released from the nanoparticles is highly dependent upon the species of serum used and the pH to which it was exposed, and in vivo studies validated the approach's predictive capability.


Viewing the selection of active siRNAs as a key limitation to RNAi drug development, scientists from the University of Oxford have created a new mouse model to study siRNA pharmacodynamics.

Current in vivo methods to determine an siRNA's biological activity are dependent on the molecular target in question, according to the researchers. "For example, where siRNA targets mRNA encoding specific enzymes, pharmacodynamics can be assessed by measuring inhibition of enzyme activity."

However, these approaches can be highly invasive and in principle require that every target-expressing cell type in the body must be evaluated to know whether siRNA activity is restricted to the intended target. Transgenic reporter mice or disease models that ubiquitously or selectively express reporter genes have been used to overcome this limitation, but each have their own shortcomings.

As such, the research group developed a positive-readout pharmacodynamic transgenic reporter mouse model that combines a luciferase reporter gene under the control of regulatory elements from the lac operon of Escherichia coli to allow noninvasive real-time assessment of siRNA activity.

"Introduction of siRNA targeting lac repressor results in increased luciferase expression in cells where siRNA is biologically active," they wrote in Molecular Therapy — Nucleic Acids.

"Five founder luciferase-expressing and three founder Lac-expressing lines were generated and characterized," they noted. "Mating of ubiquitously expressing luciferase and lac lines generated progeny in which luciferase expression was significantly reduced compared with the parental line."

Administration of lactose or a synthetic analog increased luciferase expression and fell rapidly when withdrawn in several test animals. Intraperitoneal administration of siRNA targeting lac in combination with a lipid transfection reagent increased luciferase expression in the liver, while control siRNAs had no effect, according to the team.

The researchers stated that their method is limited by interanimal variation and that additional work is required for its widespread practical application. However, "we believe a dynamic, rapidly responsive positive-readout reporter model for siRNA pharmacodynamics has great potential for improving siRNA vector design and testing accelerating the development of a new wave of genetic medicines to treat a range of disorders," they concluded.


In order to help enhance the analysis of microRNA/target interactions and help improve the overall understanding of the small, non-coding RNAs, researchers from the Free University of Berlin have developed an approach for the sequential mutation of multiple targets sites within specific 3' UTRs.

The method is based on seed mutagenesis assembly PCR, or SMAP, which allows for rapid identification of miRNA target sites, and was used to validate the transcription factor NKX3.1 as a target of miR-155. The sequential mutagenesis of multiple miRNA target sites was then examined by miR-29a-mediated CASP7 regulation, which "revealed one of two predicted target sites as the predominant site of interaction," according to a report in PLoS One.

Because the 3' UTRs of non-model organisms are either unavailable or only computationally predicted, the scientists developed stem-loop 3’ UTR RACE PCR, or SLURP, to generate required 3’ UTR sequence data. "The stem-loop primer allows for first strand cDNA synthesis by nested PCR amplification of the 3’ UTR," they noted in their paper.

Sequential seed mutation of miRNA targets using SMAP allows for rapid structural analysis of several target sites within a given 3’ UTR, and its combination with SLURP enabled the targeted analysis of miRNA binding sites in unknown mRNA 3' UTRs in a matter of days.


To further demonstrate the potential of a newly developed class of siRNAs, investigators from Japan's National Cancer Center Research Institute and Bonac Corporation have published details about a delivery approach that allows the oligos to enter the lungs and knock down cancer targets.

The RNAi molecules are synthesized as single-stranded RNAs that self-anneal into a "unique helical structure containing a central stem and two loops following synthesis," the scientists wrote in Scientific Reports.

The team created unmodified RNAi molecules targeting ribophorin II — a regulator of drug resistance in several cancers — and delivered them via aerosol into the lungs of mouse xenograft models of lung cancer. They observed an inhibition of tumor growth without any apparent toxicity.

Although additional work is required to build upon these findings — including testing the approach in multiple lung cancer cell lines and against other gene targets in animals with intact immune systems — the findings suggest that naked RNAi molecules delivered by aerosol may have therapeutic potential with minimal off-target effects.

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