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
http://tinyurl.com/256qd



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.

http://update.bmn.com/rsearch/section/record?uid=UPDATE.Thomas2901200492

Posted by
Robert Karl Stonjek.
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