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In Its Third Phase, the RNAi Consortium Looks to ncRNAs, Gene Over-Expression

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Despite its relatively low profile since it kicked off its third phase last spring, the Broad Institute's RNAi Consortium is proceeding apace, although its mission has expanded beyond its initial focus of just RNAi.

In 2004, Broad established TRC as a public/private consortium of RNAi researchers to develop genome-scale sets of virally expressed shRNAs targeting mouse and human genes (GSN 4/9/2004).

During its first phase, it created a library of about 160,000 shRNAs against roughly 32,000 human and mouse genes, and developed methods to use the library for loss-of-function genetic screens. In 2007, TRC entered its second phase, a four-year initiative focused on improving the power of that library as a screening resource by, among other things, expanding and validating the shRNA collection while refining its screening methodologies.

As reported by Gene Silencing News in 2010, TRC began making preparations for its next iteration as its second phase neared its end, deciding that it would expand its focus into non-coding RNAs, although the exact nature of that work had yet to be defined at that time (GSN 8/5/2010).

Unlike with TRC's first two phases, the direction of the third grew out of discussions between members over an extended period of time, resulting in a less publicized start — although the TRC's mission remains the same, David Root, a Broad researcher and TRC project coordinator, told Gene Silencing News last week.

“We are hoping to produce a lot of valuable know-how and resources that will be useful in the scientific community,” Root said. However, the so-called TRC3 is exploring a variety of gene-modulation technologies, not just RNAi.

“At the time we started the consortium … we were very focused on a technology that was quite new and was in great need of further expanded and characterized tool sets,” Root noted. “That's not a job that may ever be done, but we're pretty good at using it now at a certain level.”

As such, TRC3 is working with gene over-expression technologies, as well as ncRNAs such as long intergenic non-coding RNAs, or lincRNAs, and microRNAs.

“We'll continue to pursue RNAi, but we're certainly alert and interested in other approaches that might be very complementary,” he added.

In the area of over-expression, TRC3 had begun work even before its official launch in April 2011, focusing on building a library of human open reading frames for use in gain-of-function experiments.

Last June, TRC3 and collaborators published a paper detailing the production of a sequence-confirmed clonal collection of more than 16,000 human ORFs encoded in a novel vector system (GSN 6/30/2011). Using the collection, they created a “genome-scale expression collection in a lentiviral vector, thereby enabling both targeted experiments and high-throughput screens in diverse cell types,” they wrote in Nature Methods.

Currently, TRC3 is putting the ORF library into a novel lentiviral vector, “so we have an alternate route of expression,” Root explained, adding that the new collection will be barcoded to facilitate its use in pooled screens.

“You could do pooled screens using the ORF as a barcode, but of course those are of heterogeneous length and composition,” he said. “So we'll have a second complementary expression library with new capabilities.”

Within the field, TRC3 is focusing much of its attention on lincRNAs, and is in the process of creating a collection of shRNAs targeting them. The consortium already created a limited set of such shRNAs, and about a year ago TRC and collaborators from the Massachusetts Institute of Technology and Harvard University published a paper describing their use to conduct loss-of-function studies in mouse embryonic stem cells.

They found that inhibiting lincRNAs had “major consequences on gene expression patterns,” that they are “regulated by key transcription factors, and that lincRNA transcripts bind to multiple chromatin regulatory proteins to affect shared gene expression programs.”

Although it hopes to create additional lincRNA-targeting shRNAs, “we're still feeling our way as to what is the most useful reagent set for now,” Root said. “There is some evidence that a lot of non-coding RNAs have very tissue- and timing-specific expression … [but] we don't want to focus our efforts on things that are not screenable.

“Our objective is to make those things that we think multiple investigators and our members would all have some interest in screening and testing.” To that end, TRC3 is also actively developing a library of shRNAs targeting miRNAs, beginning first with human ones and potentially expanding into mouse.

Even though it has expanded beyond RNAi to new areas of genomics, TRC3 remains committed to the gene-silencing technology, Root stressed. Not only does the lincRNA and miRNA work cross over into RNAi, but the consortium is also now working with its members to enable in vivo RNAi screening.

For example, last August members of TRC3 reported in Nature on the development and use of a method to identify novel cancer targets with a negative-selection RNAi screen in a human breast cancer xenograft mouse model.

“There is more to learn about the various ways … we can use shRNAs in mice,” Root said. As its members become increasingly interested in in vivo RNAi, “we're continuing to explore … more tactical options for how to do that.”

Sponsors of TRC3, which is set to run until April 2015, include the Broad, Bristol-Myers Squibb, and Johnson & Johnson. Consortium members include Massachusetts General Hospital researcher Nir Hacohen; Dana-Farber Cancer Institute's William Hahn; Broad's Eric Lander; and MIT's David Sabatini.

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