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2-Plants: Gene silencing by RNAi - the new hype in biotechnology

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                                  PART I
-------------------------------- GENET-news --------------------------------

TITLE:  Gene silencing could wipe out farm pests
SOURCE: The New Scientist, UK, Rachel Nowak
DATE:   Mar 3, 2003

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Gene silencing could wipe out farm pests

A genetic trick known as gene silencing could help wipe out serious farm
pests such as the Mediterranean fruit fly and pink bollworm.

The release of sterile males has played a key role in the eradication of
some pests, helping eliminate the tsetse fly from Zanzibar, for example.
But the method is costly and the radiation used to sterilise the insects
can weaken them, so vast numbers must be released to ensure that wild
males do not get a chance to mate.

That has led researchers to look at ways of genetically engineering
insects to make them sterile or pass on a gene that kills all their
offspring. Now Steven Whyard of CSIRO Entomology in Canberra, the insect
division of Australia's national research institute, has shown that gene
silencing, or RNA interference, can do the trick.

RNA interference involves creating "short interfering RNAs" (siRNAs) that
combine with a gene's messenger RNA and stop it from conveying
instructions to the protein-making machinery. Plants and animals use RNA
interference to block viral genes.

Sperm switch

Whyard created an siRNA that disables a fly gene essential for sperm
development, called boule . Crucially, he linked the gene coding for the
siRNA to a genetic switch that is turned off by an antibiotic. So when
Whyard added the gene to the fruit fly Drosophila, the creatures still
bred normally when fed food laced with the antibiotic.

To create sterile males, the flies were simply taken off the antibiotic.
Of the three strains created, all the offspring were sterile in one line,
77 per cent in another and just 21 per cent in the third.

Whyard's experiments show the "repressible sterility" method is a viable
alternative to using radiation to create vast numbers of sterile males.
The technique does not solve another big problem with producing sterile
males - getting rid of the females. But other groups are trying to find
ways to genetically modify flies to prevent any females developing.

Multiple targets

For large-scale production, the team envisages using an alternative to
antibiotics to control the siRNA gene, to avoid encouraging resistance in
bacteria. And while it does not matter too much if just a few insects in
a vast batch are fertile, Whyard envisages introducing multiple siRNAs
targeting different genes.

"There are several genes besides boule that affect fertility," he says.
"We would need to target several of them so there was always a back-up."

Indeed, he thinks the technique could even be used to control genetically
modified animals such as salmon, so they could breed in captivity when
fed the right food but would become sterile if they managed to escape.

The American company Aqua Bounty, which has applied for a licence to sell
the eggs of GM salmon to farmers, currently plans to make them infertile
using a pressure technique.

The CSIRO research was presented at the Lorne Genome Conference,
Victoria, Australia

                                  PART II
-------------------------------- GENET-news --------------------------------

SOURCE: Australian Biotechnology News, by Graeme O'Neill
        edited and sent by Agnet, Canada
DATE:   Feb 28, 2003

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Who invented hairpin gene silencing? It's literally a $64 million
question, as the claimants to one of the most valuable biotechnology
patents of the past decade begin skirmishing over its ownership.

Hairpin gene silencing, also known as RNA interference (RNAi), is a
simple, quick, but exquisitely precise way of exploring gene function in
higher organisms - it can be applied to switching off genes in plants,
animals, insects, or any other higher organism. Changes in the organism
then provide cues the function of the silenced gene.

There are four contenders: CSIRO Plant Industry, the multinational life
science company Syngenta (UK), the Carnegie Institute in the US, and a
partnership involving small Queensland-based biotechnology company
Benitec Australia and the Queensland Department of Primary Industries (QDPI).

CSIRO lays claim to having discovered hairpin gene silencing in 1994;
CSIRO Plant Industry molecular geneticist Dr Peter Waterhouse performed
the first successful gene-silencing experiment in a plant - tobacco - in 1995.

CSIRO's claim rests on its belief that Waterhouse's experiment was also
the first demonstration of this novel method of gene silencing in any

But CSIRO delayed lodging its patent until April 1998; the Benitec/QDPI
partnership and Syngenta filed in March 1998. In the intervening period,
the Carnegie Institute and Syngenta filed their applications - Carnegie
filed on the basis of demonstrating hairpin gene silencing in the
nematode Caenorhabditis elegans.

Dr Rob Defeyter, a member of Waterhouse's team, and now IP manager for
Plant Industry, said the division prepared a draft patent in 1995, but
delayed filing while it conducted further research.

"Our laboratory notebooks confirm we continued our research between 1995
and 1998, mainly to confirm our data, extend the technique, and find out
how broadly applicable it might be," Defeyter said.

Benitec's claim is based on work done by former Plant Industry geneticist
Dr Mick Graham, who worked with Waterhouse until 1995 before joining QDPI.

Graham was the first to demonstrated successful dsRNA silencing in
mammalian cells, and the Benitec/QDPI alliance was the first to file a
patent on the technique, in March 1998. The question of ownership is
complicated by different views of what constitutes priority. Defeyter
said patent offices in most countries, including Australia, based
priority on the filing date, but the US Patent Office bases priority on
the date of invention - even if the patent application is not lodged
until later.

Yesterday CSIRO announced it was releasing the high-throughput gene
silencing vectors it has developed for its own work in plants, or free
use by any not-for-profit research organisation.

Today, Benitec announced a collaborative agreement with US biotechnology
company Tranzyme, in North Carolina, to develop and market its own high-
throughput gene-silencing and gene-delivery technologies.

In their joint press release, the companies said the combination of
Benitec's High Throughput (HTP) gene silencing technology announced on
January 7, 2003, with Tranzyme's industry-proven gene delivery and
expression technology, would "offer customers a revolutionary means of
producing gene 'knockdowns' quickly and efficiently.

"The combined technologies will be ideal for both in vitro and in vivo
applications," it said. "The collaboration will target the pharmaceutical
industry and will fast-track the discovery and validation of drug
targets." The companies will share revenues, as well as developing their
own proprietary targets using the combined technology for future joint

CSIRO's press release yesterday said its gene vectors can be used to
identify the function of thousands of genes quickly and accurately, and
predicted the technology would promote "major advances in biotechnology
and agriculture".

Plant Industry Chief Dr Jim Peacock said, "Scientists can now accurately
and rapidly identify the function of single genes or specific groups of
genes from tens of thousands of genes in an organism." "As well as the
speed of analysis, these vectors provide nearly 100 per cent efficiency
in 'switching off' any gene under investigation. This means the effect of
a gene in an organism can be determined confidently, and once its
function is known we can decide how to use this information." Harbinger
of attack Waterhouse's original 1995 experiment confirmed the existence
of plant cells of an ancient mechanism, perhaps evolved as a defence
against viruses, that detects the presence of double-stranded RNA
molecules - a harbinger of viral attack.

When viruses infect living cells, they produce a double-stranded RNA
(dsRNA) copy of their genetic blueprints as a prelude to mass-replicating
new virus particles.

The anti-viral mechanism detects the dsRNA genetic blueprint, and by an
as yet unidentified mechanism, cleaves it into useless fragments, halting
the virus replication before it can begin. It is now clear that the cells
of higher organisms use the same dsRNA technique to regulate their own
gene activity, by producing 'mirror image' messenger RNAs that bind to
the active gene's messenger RNA, forming a dsRNA complex - which is then

The 'knockdown' technique differs from the knockout technique used to
silence genes in rodents, in that the DNA code of the original gene is
left unaltered - the silencing is performed at the gene-transcription stage.

CSIRO's vectors, which carry names like 'Hannibal', 'Kannibal' and
'Hellsgate' are essentially plug-and-play gene cassettes into which
geneticists can insert DNA sequences copied from the many messenger RNAs
of active genes in living cells.

The gene sequence, and the complementary DNA code from the non-gene
strand, are joined end-to-end, separated by a small sequence that forms a

When the vector inserts the gene construct into living cells, the
transgene produces messenger RNAs that spontaneously fold back on
themselves, by complementary base-pairing, forming a hairpin shape.

The resulting dsRNA molecule is then detected by the cell, and destroyed
- an event that also programs the anti-viral mechanism to destroy the
'real' mRNAs from the targeted gene.

'The third great revolution' Benitec's director of research and
technology, Ken Reed, said yesterday the company was confident of its
patent position - "It's a great position to have. Mick Graham was the
first, by two and a half years, to demonstrate its efficacy in animal cells.

"It's the third revolution in biotechnology - first there was recombinant
DNA technology, then PCR, and now RNA interference," he said "We knew as
soon as the world woke up to the fact that it could be used in mammalian
cells, there would be the most massive brawl in relation to ownership of
the patent.

"In recent years, once companies realized its value, everyone has been
trawling back through their research in the early 1990s for anything that
might allow them to claim priority. It's bigger than Ben Hur."