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Pharma Impressed With Cell-Based RNAi Screens, But Acknowledges Limits

This article has been updated from a previous version, which mistakenly reported that Klaus Melchers presented information on Altana's research instead of Michael Byrne.
NEW YORK (GenomeWeb News) — Pharmaceutical firms think cell-based RNAi screens are valuable drug-discovery tools, but are finding that the approach still has a number of limitations, according to speakers at Cambridge Healthtech Institute’s Molecular Medicine Tri-Conference, held in San Francisco last week.
Several speakers at the conference noted a number of advantages of cell-based RNAi screens over small-molecule screening for target identification. The reasons include the fact that most cell types are amenable to RNAi, it’s relatively easy to knock down any gene of interest, and the resulting data is extremely informative.
The approach still has some limitations, however, most notably RNAi’s well-known tendency to produce off-target effects. In addition, several speakers discussed challenges related to the cells themselves.
For example, John Reidhaar-Olson, research leader in the department of research informatics for genetics and genomics at F. Hoffman-La Roche, noted that cell-based RNAi assays are particularly prone to edge effects because the cells in the outer wells of the plates grow at a different rate than the cells in the inner wells. As a result, his group usually ignores the outer wells in most studies, he said.
Steven Haney, group leader in oncology genomics in the department of biological technologies at Wyeth Research, said that his lab has had problems with the “penetrance” of some RNAi screens, in which the level of GFP in the cells is heterogeneous, making it difficult to interpret.
After further study, Haney said his team found that the expression levels of several proteins varied significantly within cells grown in culture, meaning that the problem is not heterogeneity of the siRNA knockdown, but heterogeneity of protein expression.
The issue is an “artifact of the cell culture itself,” Haney said, perhaps due to the fact that cells proliferate faster in culture than they would under normal biological conditions. While low penetrance can ultimately “reduce the impact of the data” from an assay, Haney said that his group has found they can improve their analysis of the data by looking at single cells rather than entire wells.
Michael Byrne, director of biochemistry at Altana Pharma, discussed another challenge related to cell lines. In this case, Altana was using the LAD 2 cell line in an RNAi screen to identify kinases related to mast cell degranulation. The problem, he said, is that LAD 2 is “a difficult cell line to work with” and “notoriously difficult to transfect.”
While the Altana team was able to account for these challenges in its assay, siRNA transfection caused a very strong interferon stress response in the cells – an unexpected side effect, Byrne said.
While the researchers first feared that this effect might be a “showstopper,” Byrne said that after further study, they found that the effects of degranulation could be “uncoupled” from the stress response, permitting them to proceed with the assay. Ultimately, he said, they were able to identify 10 novel kinase targets “that we wouldn’t have found any other way.” 

The complete version of this article appears in this week’s issue of GenomeWeb News sister publication Cell-Based Assay News.

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