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Monsanto Animal Data Points to Safety of Consumed siRNAs, dsRNAs


NEW YORK (GenomeWeb) – As interest in using RNAi for crop modification increases within the ag-bio industry, Monsanto has released new data suggesting plant-expressed dsRNAs do not affect mammals after ingestion, even when the gene-silencing molecules perfectly match an endogenous gene within the animals.

"We are developing RNAi for various agricultural applications, so we want to support the use of RNA, and adding to the story in the literature is clearly important to us," Jay Petrick, transgenic and nucleic acids product lead at Monsanto, told GenomeWeb this week. "We envisioned this study to represent an extreme scenario — very high doses of RNA test materials deliberately designed to the mouse ortholog of a gene that provides insect control."

Given RNAi's ability to selectively inhibit the expression of target genes, a number of companies have been exploring its potential to modify plants to create desirable traits such as drought tolerance, improved nutritional profiles, and pest resistance. There are already a number of agricultural products altered with the technology, and just last month the US Department of Agriculture cleared for sale strains of RNAi-modified potatoes and alfalfa.

Also nearing the market is Monsanto's Smart Stax Pro, a strain of corn that is resistant to rootworms through its expression of widely used Bt proteins derived from the bacterium Bacillus thuringiensis along with dsRNA designed to silence a gene essential to the pests known as Snf7.

Slated for commercialization by 2017, Smart Stax Pro would be the first approved agricultural product that actually expresses transgenic dsRNAs in order to combat pests.

Against this backdrop, there has been increasing scrutiny of agricultural RNAi by advocacy groups and regulators. For instance, earlier this year both the US Environmental Protection Agency and the European Food Safety Authority held scientific meetings to explore the potential risks of dsRNA-expressing plants.

RNAi experts by and large believe that RNAi plants hold little danger for humans and other mammals that consume them, largely because of the susceptibility of dsRNAs to degrade in the digestive system and the lack of evidence that these molecules can pass through the gut in any biologically active form.

Still, Monsanto has continued to generate data to support the safety of plants incorporating gene-silencing molecules as it advances Smart Stax Pro and similar products. The company has worked with microRNA drug developer Miragen Therapeutics to refute a widely criticized report that suggested that mammals can take up dietary plant miRNAs, and in April it published data showing that plant-expressed dsRNAs rapidly degrade in soil and are unlikely to persist in the environment.

Through this latest paper, which appeared in Regulatory Toxicology and Pharmacology, Monsanto has further added to the body of evidence supporting the safety of RNAi plants, demonstrating in mice that extended ingestion of dsRNAs or siRNAs with perfect sequence complementarity to the mouse ortholog of vacuolar ATPase (vATPase) — an established target for corn rootworm control — caused no toxicity in the animals.

In the study, mice were orally gavaged daily over 28 days with doses of either a pool of four vATPase siRNAs ranging from .45 mg/kg to 48 mg/kg or a 218-base pair vATPase dsRNA at doses ranging from .64 mg/kg to 64 mg/kg. Control animals received either nuclease-free water or an RNA negative control.

All animals were examined daily for mortality and moribundity, as well as changes in body weight or food consumption. Serum chemistry, hematology, gross pathology, organ weights, and microscopic pathology were evaluated at the end of the dosing period.

For all the animals, there were no instances of treatment-related effects on mortality or any clinical observation, while body weight and food consumption was similar across both the treatment and the control groups.

Similarly, there were no treatment-related differences in serum chemistry parameters for either the siRNA- or the long dsRNA-treated mice, and the statistically significant differences between study animals that were observed were attributable to inter-animal variability. Hematology parameters, organ weights, and pathology results also showed no indication of being affected by treatment with the RNAi molecules.

Gene expression analyses showed no inhibition of vATPase in the systemic tissues of treated animals as evaluated in duodenum, ileum, liver, kidney, brain, or humerus. Statistically significant increases in vATPase expression were observed in several tissues, but deemed likely to represent biological variability and not RNAi-based gene suppression.

Expression of vATPase was found to be as much as 50 percent lower in the stomachs of certain treated animals versus controls, but it is unclear whether this was due to an RNAi effect given that it only occurred in female animals at one dose level of the long dsRNA. Additionally, this effect was not accompanied by any changes in toxicological parameters or stomach histopathology.

Efforts to reproduce this effect in a second 28-day study in female mice was unsuccessful, leading the researchers to determine that the vATPase downregulation in the first study was not biologically relevant and unrelated to RNAi treatment.

Even if the finding could have been reproduced, "we would expect that a biologically relevant change in gene expression … would somehow produce a finding in the in-life parameters of the study," Petrick, who was the lead author of the study, noted. "We didn't see any toxicologically relevant changes, hence the conclusion that this was not treatment related and non-adverse."

Overall, the study adds to the growing body of data indicating that consuming dsRNAs is safe, regardless of the genes they target.

"We're eating RNAs all the time that match our genes in foods that are considered safe based on a long history of consumption, Petrick said. "These data add to the weight of that evidence, demonstrating that we have biological barriers against ingested RNA."

As such, sequence is not a determining factor in evaluating the safety of ingested RNA, calling into question the need for toxicological studies with purified RNA to establish the food and feed safety of crops expressing RNAi molecules, he added.