Articles: RNAi-based target discovery is paying off in the clinic

  • Thread starter Robert Karl Sto
  • Start date


Robert Karl Sto

RNAi-based target discovery is paying off in the clinic 30 January 2004

Cost-effective drug discovery requires a solid basis of target discovery and validation. Until
recently, target validation relied mostly on the analysis of genetic knockout mice. 'Cleanly' wiping
out single genes is an attractive model for simulating the action of the 'perfectly' selective,
potent drug, before initiating the costly drug-screening process. However, knockout mice are
difficult to make and there is no opportunity to study dose-response effects (e.g. asking if a 20%
target inhibition would be sufficient). The disease context in which we need to test the mutation is
not always obvious, and if the gene is essential for embryonic development (which is not always an
obstacle to the gene's value as a target), there might be no mouse to study at all.

The field of target discovery dramatically changed with the finding, nearly three years ago, that
short, double-stranded RNAs (siRNA or RNAi) allow researchers to knock down nearly any gene's
expression at will. Much of the current effort, such as the two Nature Genetics papers by Sen et al.
and Shirane et al., focus on ways to generate RNAi-delivering tools to probe thousands of drugable
targets in cell-based assays. The two papers describe clever methods to construct such libraries
from 'random' cDNAs.

In another recent paper, (Brummelkamp et al.Nature 2003, 424), it was demonstrated that screening
such libraries to discover 'opportunistic' targets, as opposed to discovering targets by
painstakingly unraveling or 'reverse-engineering' entire signaling pathways, is highly successful.
Brummelkamp et al. used a library to deliver sets of siRNAs against 50 human de-ubiquitinating
enzymes in various cancer-related cellular assays. These de-ubiquitinating enzymes are known to
regulate protein stability and to play a role in signaling. One of the candidates tested, CYLD, was
unexpectedly identified as a key regulator of the NF-kappaB pathway. Skin tumours were known to
develop in patients with a mutation in CYLD, but the mode of action for this oncogene was unknown.
Based on the team's observations, these cylindromatosis cancer patients are currently being tested
in the clinic with drugs that are known to block NF-kappaB activation.

Read the rest at BioMedNet

Use of RNAi to investigate the role of Arabidopsis AIP in vivo. 29 January 2004

Proper actin dynamics are required for the growth and division of cells. Many actin binding
proteins (ABPs) are involved in the regulation of these dynamics (e.g. gelsolin, AIP, ADF,
profilin). Several of these proteins have been characterized in animals, but in plant cells less is
know about the role of ABPs.

It is known that some of these ABPs work synergistically. The paper by Ketelaar et al. describes
work done on Actin Interacting Protein 1 (AIP1), which is known to cap the barbed ends of F-actin
filaments and to enhance the activity of Actin Deploymerising Factor (ADF) in vitro. They
investigated what happens when they used RNAi to remove this protein from Arabidopsis.

An RNAi construct against a conserved region of the two AIP1 Arabidopsis genes was linked to an ethanol-
inducible promoter and transformed into Arabidopsis by floral dipping. Lines showing a reduction in
the AIP1 protein (done by immunoblotting) were selfed and the T2 examined further. Three weeks after
germination, transformants were induced to express the RNAi by feeding with ethanol and the effect
on the growth and development of the plants was followed.

The results showed that growth of leaves, shoots and flowers was reduced - in some of the more
severe phenotypes the plants failed to shoot and flower and the leaves died. Root growth was also
reduced, as was the density of root hairs. The reduced size of the plants was determined to be
primarily due to reduced cell expansion rather than to reduced cell division. The authors also
studied actin organization using a GFP-FABD protein fusion. In expanding cell types of the RNAi
plants, the actin was arranged into thicker and more-compact bundles that wild-type; in root hairs,
actin bundles extended into the tip, which is not usually the case in wild-type root hairs.

Posted by Robert Karl Stonjek.