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16 April 2015AmericasCharles Brabin

Toxic traces of autumn: will exDNA make it from the forest floor to the pharmacy shelf?

The bright reds, ambers and yellows of autumn leaves are as transient as they are beautiful. Yet recent research suggests the dying leaves may have significant ecological effects that are much longer-lasting than the short-lived colours of the season.

As leaf litter decomposes, DNA is released. This extracellular DNA (exDNA) accumulates in the soil, where it appears to mediate a mysterious phenomenon that has baffled ecologists for decades: autotoxicity, whereby organisms modify their environment in a way that exerts negative effects on the growth of their own species.

Apart from solving a long-standing ecological riddle, these findings could have huge implications for agriculture and pharmaceuticals: exDNA could prove a powerful and specific tool for interacting with biological systems.

Many fundamental questions have yet to be answered but the findings are reminiscent of the discovery of a revolutionary gene-silencing technology: RNA interference (RNAi). If exDNA lives up to its expectations, a look at the dynamic world of RNAi intellectual property may provide a hint of things to come.

Co-existence of species

Ecologists have known for more than a decade that plants modify their immediate environment in a way that makes conditions increasingly negative for members of their own species. The phenomenon is widespread and plays a key role in structuring communities of plants; this ‘negative plant-soil feedback’ (NF) effect prevents dominance by one species and instead facilitates the co-existence of multiple different species.

The mechanism underlying this pattern, however, has remained mysterious.

The ecological and agricultural significance of NF has prompted much speculation and research on what might be the causal factors. Exhaustion of soil nutrients and the build-up of pathogens have both been seriously considered as explanations but do not appear to account for the patterns observed.

Another possibility is that as leaf litter decomposes it releases compounds into the soil that are toxic to members of the same species; accumulation of these compounds creates negative conditions for growth. Yet this concept of ‘autotoxicity’, too, seemed unlikely.

The toxins studied were all found to degrade rapidly in the soil, making it hard to see how they could account for long-term NF effects. Nor did the toxins have species-specific effects; rather, they exhibited general toxicity, which could not account for the specificity of NF observed in the field.

It looked as though the autotoxicity hypothesis was yet another dead end, but not all scientists abandoned the idea: in looking to either confirm or reject the concept of autotoxicity for good, a group of researchers decided to turn their attention to an alternative type of ‘toxic’ compound—DNA.

As the ‘molecule of life’, DNA perhaps seems an unlikely candidate for a growth-inhibiting autotoxic substance. Yet it attracted attention as a contender due to its abundance, its stability and its potential for highly specific interactions. ExDNA originating from decomposing leaves could survive in the soil much longer than the less stable toxic compounds investigated in other studies and, through its specificity, could have targeted rather than general toxic effects.

In a series of experiments, the researchers produced results that suggest not only that autotoxicity occurs, but that it is indeed mediated by exDNA. Application of self exDNA (DNA extracted from the same species) caused significant decreases in plant root growth, while application of non-self exDNA had no impact.

Long after the colours of autumn have faded, it seems, exDNA is released into the soil as the fallen leaves decompose. In the ground the exDNA exerts strong negative, species-specific effects on nearby plants. So striking were the results that the scientists moved on to ask whether the same principles apply outside the plant phylum.

When experiments were conducted with a diverse range of taxonomic groups—fungi, algae, protozoa and insects—the same results were obtained: treatment with self exDNA caused inhibition of growth, whereas non-self exDNA had no such effect.

The results of these experiments raise a number of intriguing questions. What is the mechanism by which exDNA exerts its negative influence? How specific is the effect? Can individual genes be selectively switched off? How widespread is the phenomenon of autotoxicity and what is its ecological significance?

Do the same principles apply to bacteria (the results of the research were less clear in this respect, with both self and non-self exDNA inhibiting bacterial growth)? The answers will all be relevant to a further key question: does exDNA represent a new and powerful tool?

A new route of intervention

The never-ending quest for new pharmaceutical products involves huge investments of time, money and research effort. The process is inefficient, with only a small percentage of candidate molecules tested reaching the market.

Among the challenges is the need for specificity of action, the evolution of resistance and, particularly in the case of agricultural products, the minimisation of environmental contamination.

As set out in patent application WO 2014/020624, the researchers behind the recent work on exDNA believe that the autotoxicity concept could provide a new route of intervention in biological systems.

“For a treatment targeting a given organism, the ‘active ingredient’ would simply be the total fragmented genomic DNA of that organism.”

As a biological application, exDNA appears to have the potential to side-step some of the problems associated with traditional drug screening and development.

Whereas synthetic or naturally-derived compounds are subjected to extensive screening processes, the self-DNA autotoxicity findings point to a simpler approach. For a treatment targeting a given organism, the ‘active ingredient’ would simply be the total fragmented genomic DNA of that organism. Specificity of exDNA appears to be high and, as a molecule present in all life forms, the harmful effects often exhibited by traditional drugs or pesticides should not pose a problem.

Further, the researchers suggest, target organisms will find it much harder to develop resistance to exDNA-mediated autotoxicity. Because the treatment would use whole genomic extracts, it has the potential to result in a wide array of genes being affected at once, making it difficult for organisms to ‘evolve around’ the intervention.

To biologists, the exDNA autotoxicity concept will have a familiar ring to it. As a system for specifically interacting with biological systems through the use of genetic material, exDNA autotoxicity lends itself to comparisons with another area of life sciences research: RNAi.

In 2006, US biologists Andrew Fire and Craig Mello were awarded the Nobel Prize in Physiology or Medicine for their discovery of gene silencing by RNAi, a natural mechanism that can be exploited to easily, efficiently and specifically switch off genes in living organisms. In order to reduce expression of a specific gene in a given organism, small interfering RNA (siRNA) molecules that match part of the sequence of that gene can be introduced to the organism. The siRNAs trigger the RNAi pathway which, in turn, causes the gene to be switched off.

The research was conducted in the late 1990s and focused on the nematode worm, Caenorhabditis elegans, but revealed a mechanism that operates across animals, fungi and plants. The discovery had an enormous impact on basic science research but was also seen as having great potential for therapeutic applications.

Gene expression—the way in which genes are turned on or off in time and space—and the correct functioning of products encoded by genes are essential for normal development. Numerous diseases are caused by inappropriate activity of genes, or by genes producing a protein that functions incorrectly. RNAi offers a mode of intervention whereby expression of the problematic gene can be minimised.

Commercial realities

For all the optimism about their therapeutic potential, RNAi-based drugs have yet to reach the market. Despite progressing to trial stages in some cases, potential treatments have encountered obstacles, particularly in the context of adverse immune reactions and difficulties delivering the molecules of RNA to target tissues.

It is not just the research and development of RNAi products that has been far from straightforward: the IP associated with this relatively new technology has also proved a complicated and dynamic area characterised by patent thickets and litigation.

Fire and Mello’s research gave rise to a fundamental patent, US number 6,506,559, known as the Carnegie patent, which covers the process of down-regulating genes through RNAi. However, while the Carnegie patent is broadly available for licensing, it became apparent that it contains significant limitations in the context of RNAi therapeutics.

The patent covers molecules of RNA that are 25 or more base pairs in length. Researchers found that siRNA molecules of this size are liable to cause dangerous immune responses, exactly the type of side effects that RNAi treatments were hoped to avoid.

Subsequently, it became apparent that using shorter fragments of RNA did not lead to the same problem. The result was a further set of patent families, Tuschl I, Tuschl II and Tuschl III, named after Thomas Tuschl, one of the researchers involved in their development. Disputes over ownership issues and exactly what the Tuschl I and II patents cover has led to multiple rounds of litigation on both sides of the Atlantic.

The sale last year of pharmaceutical company Merck’s RNAi portfolio, which included the Tuschl I patents, to Alnylam, exclusive licensee of the Tuschl II patents, may signal a period of relative calm.

However, the sale also demonstrates the volatility of the RNAi therapeutics market—what they sold in early 2014 for $175 million they had bought for $1 billion in 2006 when acquiring Sirna Therapeutics.

Following Roche’s decision to move out of the RNAi field in 2010, the Merck sale serves as a reminder that, despite the promise of huge potential, even the largest of pharma firms have their reservations about committing to what is still an emerging field. Yet RNAi therapeutics have by no means been abandoned: multiple treatments for cancer and viral diseases continue to progress through testing pipelines.

Delivery remains one of the biggest challenges for the field and several companies are focused on developing different ways of delivering siRNAs to target cells: use of lipid nanoparticles to transport the molecules through the bloodstream and into target cells; conjugating siRNAs to sugars that bind specifically to certain cell types; and using modified viruses to inject the siRNA into patients’ cells. Together with RNAi systems that target specific genes, delivery approaches look set to be an area of increasing IP activity.

Despite past setbacks, RNAi is widely expected to yield useful therapeutic treatments in the coming years. As long as the development of new systems and treatments remains a research focus, RNAi IP estates will continue to be of value through their potential to generate income through licensing fees.

As and when products near the market, this value, along with the licensing fees and, perhaps, the likelihood of further disputes, will increase.

Excitement about potential therapeutic applications of RNAi has led to vast numbers of patent applications and numerous prolonged IP disputes.

The field of exDNA is still nascent and it is far from clear whether the proposed applications of this newly discovered system will be realised; considerable further research will be required to deal with the numerous fundamental unknowns that hang over the latest findings. However, if exDNA does live up to its expectations, the IP associated with its applications may well be an active area in the coming years.

Charles Brabin has a DPhil in genetics and is a non-practising barrister. He can be contacted at: charles.brabin@magd.oxon.org

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