BIO 2009 Through the glasshouse: Hexima’s stylish resistance Agbiotech often sails under the radar, but company Hexima has been making waves with its fungal and insect-resistance technology, and with its deal with Pioneer Hi-Bred International, the agricultural business unit of multinational DuPont. Kate McDonald reports.

It was serendipitous to say the least the company’s scientific team and heads the that just a month after he took up the role company’s research and discovery based at as CEO of listed agbiotech Hexima in July ; and Dr Robyn Heath, 2008, Josh Hofheimer was able to announce who established the company’s cotton trans- publicly a terrific deal with agriculture formation capability and field trials, and is giant DuPont. That deal involves the mul- now head of Hexima’s product development tinational transferring exclusive control of team based at the . its transgenic anti-fungal protein disease These scientists, along with Dr Angela research to a small Melbourne company Atkinson, published a series of ground- that started out researching self-incompat- breaking papers in the 1980s and 1990s that ibility in plants. form the basis of the technologies now being Under the deal, with DuPont’s Pioneer commercialised by Hexima. Hi-Bred International business, DuPont took The IP involves three technologies: a five per cent stake in Hexima, which in turn the fungal-resistance technology based on received exclusive commercialisation rights defensin proteins; insect-resistance technol- Joshua Hofheimer to Pioneer’s library of anti-fungal proteins. ogy based on proteinase inhibitors (PIs); and The companies have combined their a multi-gene expression vehicle (MGEV) that This is a question that has fascinated respective libraries and Hexima is now respon- does what the name suggests – allows scientists plant biologists since the days of Darwin, sible for identifying and developing anti-fungal to deliver multiple proteins to a particular Clarke says. “This is a very important gene in resistance traits, while DuPont will take on cellular site from the same transcript. plants, of course, because the ability of plants later-product development and marketing of to recognise and reject their own pollen was a those traits in corn and soy. Women’s business critical event in the evolution of plants. It’s not the first time Hexima has worked Hexima was founded in 1998 but existed “If they couldn’t reject their own pollen with a multinational – it is coming to the end of as a virtual company until it was spun-out then they’d have incestuous mating and the a contract with Dow AgroSciences to perform by the University of Melbourne in 2001 and problems of inbreeding depression. Then of cotton transformation experiments – but it is the current group of major shareholders course they wouldn’t flourish and we wouldn’t a stamp of approval for both the company and invested. The company listed in August be here today.” its technologies. And in Hofheimer’s words, 2007, raising a tidy $40 million and achiev- Clarke’s group received a large grant from Hexima is one of the few small-cap biotechs in ing an initial market capitalisation of $100 the Commonwealth and another from the US the world that is taking its research “through million. company Agrigenetics in the 1980s to fund the the glasshouse and into the field” in com- It is not travelling so high now – few bio- cloning work. “With the beginning of the revo- mercial crops. techs are – but it has excellent cash reserves lution in molecular biology, we thought the “A multi-billion company is going to of almost $32 million and a spend of only time was ripe for a molecular understanding take its technology, its lengthy investment $7-8 million per annum, which should see of the basis of self-incompatibility,” she says. in genetic anti-fungal protein disease resist- it through another four years of R&D. The Fellow carbohydrate chemist and enzy- ance and combine that and give exclusive company even generates revenue through its mologist Marilyn Anderson, a graduate of control over that technology to a $30 million contract with Dow. the University of Melbourne and La Trobe Australian company,” he says. “That DuPont It is also seeking to commercialise its University, had by then returned from Cold has entered such partnership with Hexima MGEV and PI technologies, and has also Spring Harbor Laboratory and brought her attests to the stature of our scientific team begun construction of a new glasshouse, skills in molecular biology to the project of and our technology.” complete with tissue culture facilities and cloning the self-incompatibility gene from That scientific team includes Professor robotics, at La Trobe University. Nicotiana alata. Adrienne Clarke, one of ’s best-known This is all a far cry from the 1980s when They managed to clone the gene – known scientists and former chair of the CSIRO, Adrienne Clarke headed the Plant Cell as the S-gene and one of the most widespread who is Hexima’s chief scientific advisor and Biology Research Centre at the University of of plant self-incompatibility mechanisms – deputy chair; Professor Marilyn Anderson, Melbourne, where her team set about cloning by looking at the proteins associated with who discovered the company’s insecticidal the gene that controls self-incompatibility and anti-fungal molecules and now leads in plants. Continued on p30 >>

28 March/April 2009 aUSTR ALIAN lIFE sCIENTIST www.lifescientist.com.au BIO 2009 Victoria

<< Continued from p28 bacterial attack. And of course it does – the plant version of the defensin protein family. specific alleles. The gene turned out to code One easy experiment was to inoculate the for a series of ribonucleases expressed in surface of the stigma with fungi or bacteria. If the plant’s style, which arrest the growth of the innoculum seeps on to the petals or leaves, the pollen tube. they are quickly infected and the tissues are This work was published in a series of destroyed, but the stigma and style remain papers in Nature in the 80s and 90s, and along intact and undamaged. the way the team began cloning some of the Defensins are part of a very large family other genes associated with female sexual of molecules, but Hexima has managed to tissues. One of these was a gene in Nicotiana identify several that are very highly expressed alata that was highly expressed in the female and very potent against fungal disease. The stigma and coded for inhibitors of the digestive scientists engineered these into cotton, which enzymes trypsin and chymotrypsin. at that time was the only GM crop allowed to “But the protein in the stigma didn’t pre- be grown in Australia, with good early results. cisely match the gene,” Clarke says. Robyn The team set up a cotton transformation Heath was then a PhD student with Anderson, system and then moved into field trials. This and she and another student, Angela Atkinson, is what first attracted the attention of DuPont, endeavoured to find out what this gene, later which was interested in the technology’s poten- called NaPI, really coded for. It turns out tial in corn. Hexima is now setting up a corn that it codes for a 42 kD protein which is Adrienne Clark transformation system, with the assistance then processed to give six separate proteinase of DuPont scientists. inhibitors: two chymotrypsin inhibitors and The company has conducted two years four trypsin inhibitors. Resistance with style worth of field trials of the defensin technology “Marilyn’s team then discovered that the Nicotiana alata, the common ornamen- in cotton against Fusarium wilt and one year protein circularises, so the ends are folded tal tobacco plant, has proved an excellent against Verticillium wilt, and a third year of around like a clasp,” Clarke says. “We describe model to work with for the Hexima scien- cotton disease trials are underway. Under it as having the proteinase inhibitors being tists. It has very well-defined self-incompat- the deal, DuPont has exclusive rights to the linked like beads on a string, like a bracelet, ibility, and the female stigma and style are technology in corn and soy, while Hexima and the clasp holds the bracelet together with easy to collect. retains rights to the other crops. three disulphide bonds. The plant then proc- Female sexual tissues are at the heart of Early trials with the PIs have been promis- esses this and clips back the linker sequences Hexima’s science. When the team was in the ing, but not quite effective as a commercial to release six proteinase inhibitor units.” early stages of research into finding out what alternative to Bt. That’s not to say the next (This later gave rise to Hexima’s name, the PI gene coded for, they pondered why the generation of PIs won’t be more effective, but she says. The team was in a rush to register plant would put so much effort into producing for the time being the company is looking at the company but every name they thought such large amounts of protein in the female whether PIs can be combined with Bt to make of had already been bagged. “It was about stigma, Clarke says. an overall more effective product. seven o’clock at night and our lawyer said we “Why was it so? We thought it could be to “We have identified additional proteinase had to come up with a name that night. So I protect the female tissues from insect attack. inhibitors and using the MGEV as an expres- mentioned hexamer, as in monomer, dimer, On the one hand, the stigma surface is covered sion tool, we plan to combine multiple PIs and trimer etc. Being a lawyer, he just wrote it with a sticky secretion to enable efficient cap- create a more robust trait, which is important phonetically, so we became Hexima.”) ture of pollen. On the other hand, this secre- for resistance management and for refuge This work forms two of Hexima’s tech- tion could be a good food source for insects or management, and potentially for improving nologies: the proteinase inhibitor technology a good substrate for fungi or bacteria. the spectrum of the trait,” Hofheimer says. for insect control and the MGEV. Anderson “Yet in the field, the stigma of flowers “We think there is the potential to be a collaborated with Professor David Craik from rarely suffers from insect attack or infection. stand-alone product. However, when you the University of Queensland, a world expert Our question then was, are the proteinase combine the PIs with Bt you may have an in NMR spectroscopy and protein structure, inhibitors involved in protecting the female improved product than from Bt alone. The to examine the proteinase inhibitor ‘beads tissues from insect attack?” advantage is that you get multiple modes of on a string’. The team discovered that when the protei- action and potentially expand the spectrum “Marilyn’s team learned that this par- nase inhibitors were fed to certain insects in of pests that you can target.” ticular structure was very flexible around the an artificial diet, the growth and development “We are exploring partnership opportuni- linker sequence,” Clarke says. “That then led of the larvae are severely affected. “Our strat- ties to develop our insect resistance technol- to the idea that we could, within this gene, egy was then to transfer the genes encoding ogy, and we think the MGEV has value as a replace units, so you take out one or more certain of these inhibitors to crops vulnerable protein delivery vehicle that can help you get proteinase inhibitor genes encoding differ- to insect attack, such as cotton, and look for traits and multiple proteins in one location.” ent molecules. This would enable delivery of protection against insect attack,” Clarke says. “With the push towards stacking, these multiple proteins to a particular site from the If the plant was expending so much energy big companies are looking for tools that can same transcript. That became what we call protecting its eggs from insects, it would prob- help them do that stacking in a controlled our multi-gene expression vehicle.” ably have similar weapons against fungal or way.” ALS

30 March/April 2009 aUSTR ALIAN lIFE sCIENTIST www.lifescientist.com.au