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Monsanto Study Finds Quick Degradation of dsRNA in Soil

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With the Environmental Protection Agency in the midst of establishing a framework for assessing the potential risks of dsRNA-expressing crop plants, researchers from ag-bio giant Monsanto have published a report indicating that such RNAi molecules degrade quickly in soil and therefore are unlikely to persist in the environment.

The findings specifically relate to dsRNA expressed by Monsanto's soon-to-be-commercialized strain of pest-resistant corn called Smart Stax Pro and were generated as part of a standard regulatory framework Monsanto has in place to determine the environmental impact of its products, Samuel Dubelman, a senior scientists at Monsanto and study co-author, told Gene Silencing News.

Because of the dearth of information available on the fate of dsRNA in soil, the data also provide insights that can be used to evaluate future RNAi-incorporating agricultural products, he noted.

While a number of companies are exploring the potential of plants that express gene-silencing dsRNAs as a way to combat destructive insects or enhance resistance to environmental stressors, Monsanto is the farthest along in the process. Smart Stax Pro, the company's most advanced product candidate of this kind, expresses two widely used pesticidal Bt proteins along with dsRNAs against a gene called Snf7 to kill Western corn rootworms. It is set for market launch by 2017.

Amid such efforts, the EPA recently kicked off an effort to better understand the possible dangers these kinds of plants pose to humans and the environment, and in January held a meeting of agency scientists and outside researchers to solicit input on key concerns and questions surrounding the agricultural application of RNAi.

Among the EPA's biggest concerns is whether dsRNAs would enter and persist in the soil of crop fields, thereby threatening non-target organisms.

Previously, Monsanto investigators reported that the silencing effect via dsRNA against DvSnf7 — the rootworm ortholog of Snf7 — has a "narrow" spectrum and is therefore unlikely to affect other animals in the insects' environment. With the newest publication, the company has provided additional data suggesting that the window for such unintended exposure is equally limited.

"This type of study provides an additional level of comfort that, even in the unlikely event there are other organisms that could be harmed, there will be no opportunity for exposure because [the RNAi molecule] degrades so fast," Dubelman said.

In their paper, which appeared in PLOS One, the scientists noted that Bt proteins are known not to persist in soil for very long, making the environmental exposure risks minimal and leading the EPA to determine that they do not accumulate as a result of continuous cultivation.

Meanwhile, DNA has been found to persist for even shorter periods of time in viable soil than Bt proteins — around 3 days for the former versus as much as 30 days for the latter. However, "less information is available on the degradation kinetics of RNA in soil," although existing information suggest that it persists for longer than DNA, the team wrote.

But because of the likely development of new products with RNA-based modes of action, "more information is needed to understand the fate of dsRNA in the soil environment," they noted.

To gather this information, the Monsanto group tested the environmental fate of DvSnf7 in three different soils that have the physical characteristics of world regions where corn is grown, including a silt loam from Illinois, loamy sand from Missouri, and clay loam soil from North Dakota. The samples were also amended with a mixture of lyophilized root and shoot corn tissue to simulate post-harvest field environmental conditions.

Further, the soil samples were treated with a solution of DvSnf7 dsRNA to "ensure that a robust quantitation could be obtained at all time intervals of an experimental range covering at least 90 percent degradation of the initial DvSnf7 RNA concentration," they wrote in PLOS One.

During a 48-hour incubation period, samples were collected at 12 time points and studied using either QuantiGene molecular analysis or insect bioassay methods. The half-life values of DvSnf7 RNA in silt loam, loamy sand, and clay loam silt were found to be 19, 28, and 15 hours based on QuantiGene, and 18, 29, and 14 hour based on the insect bioassay, respectively.

Further, the time to 90 percent dsRNA degradation for all three soils was less than 35 hours — a relatively rapid rate when compared with Bt proteins.

The scientists determined that the dsRNA degradation is largely due to metabolism by the resident microbial community, although microbial-produced ribonucleases also likely mediate the degradation process.

"Taken together, these results demonstrate that [plant-expressed] DvSnf7 dsRNA … is unlikely to persist or accumulate in the soil environment," the team concluded. "In a broader context, these results provide fundamental information to define environmental exposure scenarios for ecological risk assessment of future RNA-based agricultural products."

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