New Therapeutic Targets
Cenix BioScience (www.cenix-bioscience.com) presented information on its RNAi-based collaboration with Bayer HealthCare (www.bayerhealthcare.com) that produced several novel therapeutic targets. "This is a large study and a nice illustration of what high-throughput RNAi screening can provide," said Christophe Echeverri, Ph.D., CEO and CSO.
"The screen I describe is in the area of atherosclerosis, identifying genes that, when silenced, cause an upregulation of LDL uptake. This is a major way to lower blood cholesterol levels."
Bayer transfered cell-based assays in five different disease areas to Cenix. The first phase of the project was to optimize these so they could be run as high-throughput RNAi screens, with the hope that many of them would reach screenable conditions over the 5,000 genes chosen to be therapeutically relevant.
The company combined its high-throughput RNAi with high-content microscopy-based readouts, which integrated three parameters: LDL uptake, cell proliferation, and transferrin uptake as a secondary control assay to make sure the chosen hits specifically affected LDL uptake.
Dr. Echeverri said that a big advantage of high-throughput RNAi screens is that they can eliminate false positives if well-designed and controlled, providing direct links between therapeutic phenotypes and individual genes. However, like all other screening methods, high-throughput RNAi screens will inevitably produce false negatives.
As an example, he described a gene in the cholesterol-screening pathway they wanted to use as a positive control. Out of five different siRNAs designed to target this gene (all provided more than 80% silencing), only the best one, providing 95% silencing, showed the expected induction of LDL uptake.
"This tells you the gene is part of that pathway, but the silencing threshold needed to detect loss of function is extremely high and tight," Dr. Echeverri added.
At the end of the screen, they had about 40 to 50 hits. "We decided that RNAi offers a great tool to identify those genes easiest to render rate-limiting. We realized we could take siRNAs targeting each of these hits and titrate down the expression of the genes by titrating the RNAi conditions. You can then relate back how much each one needs to be silenced before it starts being rate-limiting for the entire pathway," explained Dr. Echeverri.
This new validation approach is called Pathway Titration, and provides information that previously was not available. "This can be used as a new criterion for prioritizing target candidates for launching compound screens," according to Dr. Echeverri.
Adenoviruses provide high transduction efficiency and good reproducible results. This is why GE Healthcare (www.gehealthcare.com) recently launched its ready-to-use validated adenoviral gene-delivery reagents.
"We've demonstrated that by comparing this to traditional plasmid-based transient transfection techniques, the adenovirus allows you to put the gene of interest, in our case the assay sensor, to as wide a variety of cells as possible. People want to have a rapid turn-around to produce a new cell sensor," stated Stephen Game, Ph.D., technology manager, in-cell assays.
The adenovirus is simply added to the cells to quickly allow optimization of the protocol. It can be used with a wide variety of primary and transformed cell types, and the vectors allow interrogation of multiple pathways within a cell.
"We're trying to cover as many signaling pathways as possible; right now about 12 to 20 key signaling pathways are represented," added Dr. Game. "At the moment we're looking at the cytokine signaling pathway associated with statin-signaling responses."
Each Ad-A-Gene Vector contains a gene encoding a protein target fused to either EGFP (emerald green fluorescent protein) or a gene encoding a response element controlling the expression of the NTR (nitroreductase) reporter gene. The first tends to measure the earlier cell signaling in the transduction pathway, and the second follows a later stage of signal transduction.
Dr. Game says that this new gene-delivery reagent opens the door to high-throughput. "One of the things we want to do is show this is compatible with formats that can be automated. In principal, we would see these being used upstream and downstream of primary screening, like validation efforts associated with profiling against cell-signaling pathways." The company currently offers eight Ad-A-Gene Vectors. Its goal is to have 50 commercially available by the end of this year.