Patently, a battle for genetics' next big thing

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 organism.

But CSIRO delayed lodging its patent until April 1998; the Benitec/QDPI partnership filed in March 1998. The Carnegie Institute and Syngenta filed their applications in 1997 -- 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 1996 before joining QDPI.

Graham claims to have been the first to demonstrate successful dsRNA silencing in mammalian cells, and the Benitec/QDPI alliance filed a patent application 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, for 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 commercialisation.

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 in 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 degraded.

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 hinge. 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."