Delivery of Synthetic and Viral-Based Gene Silencing

The rapid evolution of RNAi-based gene silencing techniques has provided a number of tools that are being utilized in a variety of screening projects. The technology has enabled drug discovery researchers to perform loss-of-function screens within increasingly rapid time frames and in cell types that more closely resemble in vivo models. siRNA screens are more common, but have been largely limited to easily transfectable cell types. Viral-based shRNA screening strategies are still evolving, but they offer the advantage of long-term knockdown in a variety of cell types, including primary and non-dividing cells. Developing delivery solutions for both synthetic siRNA and viral-based shRNA screening is a key focus for Sigma-Aldrich.

siRNA Screening

The primary advantages of using synthetic siRNAs for screening are their ease of use and the greater availability of established delivery methods. Available siRNA libraries have been extensively validated and give robust silencing. However, delivery of siRNA into difficult-to-transfect cell types has been a hurdle for the drug discovery pipeline. Initial screens are often performed in cell types that do not closely approximate the in vivo disease model. Primary cells are more similar to their in vivo counterparts, but are also typically more recalcitrant to delivery. Potential candidates or “hits” that are discovered in these initial screens must then be assayed in primary cells where chemical delivery methods are often ineffective. The availability of a delivery reagent capable of transfecting siRNAs into primary cells could significantly decrease the time to validate hits and enable the screening of primary cells with siRNAs.

Sigma-Aldrich has developed a potent strategy for overcoming this barrier. The N TER™ Nanoparticle siRNA Transfection System enables siRNA delivery into a wide variety of adherent cells, including primary, neuronal, and non-dividing cells. The novel peptide-based nanoparticles interact directly with lipids on the surface of the plasma membrane, allowing the nanoparticles to diffuse across the cell and deliver siRNA to the cytoplasm. Most importantly, N-TER has been experimentally validated in over 20 difficult-to-transfect cell lines, such as human astrocytes, BSMC, HUVEC, and NHEK-AD, as well as fibroblasts and macrophages.

The N-TER transfection system has been coupled with the MISSION siRNA Druggable Genome Libraries and gene family panels to offer a unique screening solution.The Rosetta Inpharmatics siRNA design algorithm, used to design the libraries, was optimized over the course of 3 years with continuous development for enhanced performance. New and critical rules were incorporated to lead to increased target specificity and knockdown for low abundance messages. Design modifications around the seed region also result in reduced off-target effects. For each target gene, 3 individual siRNA duplexes with 3’-TT overhangs are designed. Panels of these siRNAs have recently been developed for human, rat, and mouse gene families for flexibility in cell type screening.

shRNA Screening

While the siRNA mediated knockdown of gene expression is transient in nature, viral-based shRNAs enable long-term silencing for more complex pathway analysis and screening. Many proteins are too abundant or stable for phenotypic changes to be assessed in transient knockdown experiments. However, with lentiviral delivery, the shRNA sequence stably integrates into the host chromosome for long-term reduction of mRNA, thus extending the assay time and allowing for the measurement of protein reduction. Unlike siRNA screens, the hurdle for shRNA screens is the availability of pre-cloned libraries. The MISSION TRC shRNA libraries were developed by The RNAi Consortium (TRC), based at the Broad Institute of MIT and Harvard. In addition to the Broad Institute, TRC partners include Harvard Medical School, Dana-Farber Cancer Institute, the Whitehead Institute for Biomedical Research, Novartis, Eli Lilly, Bristol-Myers Squibb, and Sigma-Aldrich. With the goal of developing and validating tools and methods that will enable the scientific community to use RNAi to unveil the function of most human and mouse genes, the MISSION TRC shRNA Libraries are a comprehensive collection of more than 150,000 pre-cloned shRNA constructs. Each human and mouse library contains lentiviral-based target sets, consisting of 3-5 individual clones per gene, corresponding to more than 15,000 genes.

Lentiviral systems have numerous features that make them particularly useful for shRNA delivery, including broad tropism, stable integration, and negligible interferon response upon transduction. The MISSION TRC lentiviral particles are pseudotyped with VSV-G (glycoprotein of vesicular stomatitis virus) for the broadest tropism and transduction of virtually any mammalian cell type, including terminally differentiated cells. The constructs are produced using a third generation lentiviral system for enhanced safety.

As the exclusive distributor of the lentiviral particle format, Sigma-Aldrich has become the leader in high-throughput lentiviral manufacturing. We deploy state-of-the-art robotic handling, LIMS, and quality manufacturing systems for production of individual clones, target sets, gene family sets, and entire libraries. The expert manufacturing team has also extended their capabilities to include custom, large-scale viral production for in vivo use and a variety of volumes and concentrated titers, as well as custom packaging of other libraries, to meet the needs of pharmaceutical discovery.

Conclusion

Sigma-Aldrich is partnering collaboratively with Rosetta Inpharmatics and with The RNAi Consortium (TRC) to provide you with unmatched infrastructure and service in synthetic siRNA and viral-based shRNA screening applications. More choices in design and delivery enable better RNAi performance.