Jay S. Petrick

Lack of detectable oral bioavailability of plant microRNAs after feeding in mice

965 chow preparation where we observed the expected distribution and abundance of rice miRNAs. Oryza sativa (rice) osa-miR168a was among the most abundant miRNAs (Supplementary Table 1) in both ricecontaining chow and rice grain, consistent with previous reports9,12. Diet composition had no impact on food consumption (Supplementary Fig. 1). Following completion of the feeding regimen, small RNAs were sequenced from mouse liver and plasma samples using the Illumina (San Diego) HiSeq system. Details of the experimental protocols can be found in Supplementary Methods. We observed the expected endogenous miRNA profile and miRNA length distribution in mouse plasma and liver and rice samples, indicating consistent quality of the small RNA sequencing procedure (Supplementary Fig. 2a,b). Analysis of small RNAs from plasma and liver of mice fed on balanced rice chow and rice chow did not reveal measurable uptake of any rice grain miRNAs, including osa-miR168a. Of >10 million total sequence reads per library, fewer than ten reads identical to known rice miRNAs per library were noted in five out of eight samples from mice fed on rice-containing chow and four out of five samples from mice fed on synthetic chow (Table 1). Synthetic chow did not contain any grain or forage from plants (all plant-derived ingredients were highly purified isolates, for example, cornstarch and soybean oil); therefore, these low number of rice miRNAmappable reads could be explained by sequence errors or by cross-contamination. Mapping of mouse small RNA data to all annotated rice miRNAs in miRBase v19 identified a low number of mouse small RNA reads identical to several rice miRNA sequences (Table 1). Even so, most of the rice-like sequences were identical to the miR414 sequence (Supplementary Table 1), which is not detectable in rice grain12. In addition, these plant-like sequences were present in similar quantities in all mouse To the Editor: Human therapeutics based on nucleic acid targeting rely on sequence-specific interactions between effector and target molecules to achieve beneficial effects through specific modification to the expression of targeted genes. A variety of such compounds are being tested in laboratories and in clinical trials to treat a range of genetic and acquired diseases. Efficient, safe and cost-effective delivery of nucleic acid–based drug candidates is required to enable therapeutic levels of targeted gene regulation and overall success of this exciting new class of therapeutics. For several types of compounds in this class, effective drug delivery relies on injection of formulated nucleic acids at the site of action or into the bloodstream. Oral delivery would excel as a treatment strategy as it could offer convenient and patient-friendly features, however, progress in this approach has been hampered by substantial challenges associated with biological barriers that limit oral activity of nucleic acid therapeutics (e.g., stability within and uptake of nucleic acids from the mammalian gastrointestinal tract, nucleases and membrane barriers)1. Considerable effort has been applied to improve the stability and uptake efficiency of orally administered nucleic acids by introducing chemical modifications and formulating with excipients; however, limited success has been reported thus far2,3. The naturally occurring RNA interference (RNAi) response has been extensively reported after feeding double-stranded RNA (dsRNA) in some invertebrates, such as the model organism Caenorhabditis elegans4 and some agricultural pests5,6 (e.g., corn rootworm and cotton bollworm). Yet, despite responsiveness to ingested dsRNA, a recent survey revealed substantial variation in sensitivity to dsRNA in other Caenorhabditis nematodes7 and other invertebrate species8. In addition, despite major efforts in academic and pharmaceutical laboratories to activate the RNA silencing pathway in response to ingested RNA, the phenomenon had not been reported in mammals until a recent publication by Zhang et al.9 in Cell Research. This report described the uptake of plant-derived microRNAs (miRNA) into the serum, liver and a few other tissues in mice following consumption of rice, as well as apparent gene regulatory activity in the liver. The observation provided a potentially groundbreaking new possibility that RNAbased therapies could be delivered to mammals through oral administration and at the same time opened a discussion on the evolutionary impact of environmental dietary nucleic acid effects across broad phylogenies. A recently reported survey of a large number of animal small RNA datasets from public sources has not revealed evidence for any major plant-derived miRNA accumulation in animal samples10. Given the number of questions evoked by these analyses, the limited success with oral RNA delivery for pharmaceutical development, the history of safe consumption for dietary small RNAs11 and lack of evidence for uptake of plant-derived dietary small RNAs, we felt further evaluation of miRNA uptake and the potential for cross-kingdom gene regulation in animals was warranted to assess the prevalence, impact and robustness of the phenomenon. To address this question, we conducted a well-controlled mouse feeding study with rice-containing chow diets or with a purified synthetic chow devoid of plant grain or forage. After a two-week acclimation on synthetic chow (modified AIN93-G), animals were fasted for 12 h and then placed on synthetic chow; a nutritionally balanced, rice-containing chow (modified AIN93-G with 40.8% rice); or rice-based chow (75% rice), for 1, 3 and 7 days (Supplementary Methods). These groups are referred to herein as synthetic chow, balanced rice chow and rice chow, respectively. To confirm rice miRNA availability in feeding material, we first sequenced rice small RNAs from ricecontaining chow and rice grains used for Lack of detectable oral bioavailability of plant microRNAs after feeding in mice correspondence

Analysis of plant-derived miRNAs in animal small RNA datasets

BackgroundPlants contain significant quantities of small RNAs (sRNAs) derived from various sRNA biogenesis pathways. Many of these sRNAs play regulatory roles in plants. Previous analysis revealed that numerous sRNAs in corn, rice and soybean seeds have high sequence similarity to animal genes. However, exogenous RNA is considered to be unstable within the gastrointestinal tract of many animals, thus limiting potential for any adverse effects from consumption of dietary RNA. A recent paper reported that putative plant miRNAs were detected in animal plasma and serum, presumably acquired through ingestion, and may have a functional impact in the consuming organisms.ResultsTo address the question of how common this phenomenon could be, we searched for plant miRNAs sequences in public sRNA datasets from various tissues of mammals, chicken and insects. Our analyses revealed that plant miRNAs were present in the animal sRNA datasets, and significantly miR168 was extremely over-represented. Furthermore, all or nearly all (>96%) miR168 sequences were monocot derived for most datasets, including datasets for two insects reared on dicot plants in their respective experiments. To investigate if plant-derived miRNAs, including miR168, could accumulate and move systemically in insects, we conducted insect feeding studies for three insects including corn rootworm, which has been shown to be responsive to plant-produced long double-stranded RNAs.ConclusionsOur analyses suggest that the observed plant miRNAs in animal sRNA datasets can originate in the process of sequencing, and that accumulation of plant miRNAs via dietary exposure is not universal in animals.

Application of food and feed safety assessment principles to evaluate transgenic approaches to gene modulation in crops

New crop varieties containing traits such as enhanced nutritional profiles, increased yield, and tolerance to drought are being developed. In some cases, these new traits are dependent on small RNAs or regulatory proteins such as transcription factors (TF) that modify the expression of endogenous plant genes. To date, the food and feed safety of genetically modified (GM) crops has been assessed by the application of a set of internationally accepted procedures for evaluating the safety of GM crops. The goal of this paper is to review the main aspects of the current safety assessment paradigm and to recommend scientifically sound principles for conducting a safety assessment for GM crops that are developed by technologies that modify endogenous plant gene expression. Key considerations for such a safety assessment include the following: (1) RNA and TF are generally recognized as safe (GRAS); (2) Genes encoding RNAi and regulatory proteins such as TFs are an important component of the plantgenome; (3) Crops engineered using RNAi modifications are not expected to produce heterologous proteins; (4) The modulation of TFs may result in quantitative differences in endogenous plant components,which can be assessed through agronomic performance and compositional analysis on a caseby-case basis.

Safety assessment of food and feed from biotechnology-derived crops employing RNA-mediated gene regulation to achieve desired traits: A scientific review

Gene expression can be modulated in plants to produce desired traits through agricultural biotechnology. Currently, biotechnology-derived crops are compared to their conventional counterparts, with safety assessments conducted on the genetic modification and the intended and unintended differences. This review proposes that this comparative safety assessment paradigm is appropriate for plants modified to express mediators of RNA-mediated gene regulation, including RNA interference (RNAi), a gene suppression mechanism that naturally occurs in plants and animals. The molecular mediators of RNAi, including long double-stranded RNAs (dsRNA), small interfering RNAs (siRNA), and microRNAs (miRNA), occur naturally in foods; therefore, there is an extensive history of safe consumption. Systemic exposure following consumption of plants containing dsRNAs that mediate RNAi is limited in higher organisms by extensive degradation of ingested nucleic acids and by biological barriers to uptake and efficacy of exogenous nucleic acids. A number of mammalian RNAi studies support the concept that a large margin of safety will exist for any small fraction of RNAs that might be absorbed following consumption of foods from biotechnology-derived plants that employ RNA-mediated gene regulation. Food and feed derived from these crops utilizing RNA-based mechanisms is therefore expected to be as safe as food and feed derived through conventional plant breeding.

Endogenous small RNAs in grain: Semi-quantification and sequence homology to human and animal genes

Small interfering RNAs (siRNAs) and microRNAs (miRNAs) are effector molecules of RNA interference (RNAi), a highly conserved RNA-based gene suppression mechanism in plants, mammals and other eukaryotes. Endogenous RNAi-based gene suppression has been harnessed naturally and through conventional breeding to achieve desired plant phenotypes. The present study demonstrates that endogenous small RNAs, such as siRNAs and miRNAs, are abundant in soybean seeds, corn kernels, and rice grain, plant tissues that are traditionally used for food and feed. Numerous endogenous plant small RNAs were found to have perfect complementarity to human genes as well as those of other mammals. The abundance of endogenous small RNA molecules in grain from safely consumed food and feed crops such as soybean, corn, and rice and the homology of a number of these dietary small RNAs to human and animal genomes and transcriptomes establishes a history of safe consumption for dietary small RNAs.

Safety assessment considerations for food and feed derived from plants with genetic modifications that modulate endogenous gene expression and pathways.

The current globally recognized comparative food and feed safety assessment paradigm for biotechnology-derived crops is a robust and comprehensive approach for evaluating the safety of both the inserted gene product and the resulting crop. Incorporating many basic concepts from food safety, toxicology, nutrition, molecular biology, and plant breeding, this approach has been used effectively by scientists and regulatory agencies for 10-15 years. Current and future challenges in agriculture include the need for improved yields, tolerance to biotic and abiotic stresses, and improved nutrition. The next generation of biotechnology-derived crops may utilize regulatory proteins, such as transcription factors that modulate gene expression and/or endogenous plant pathways. In this review, we discuss the applicability of the current safety assessment paradigm to biotechnology-derived crops developed using modifications involving regulatory proteins. The growing literature describing the molecular biology underlying plant domestication and conventional breeding demonstrates the naturally occurring genetic variation found in plants, including significant variation in the classes, expression, and activity of regulatory proteins. Specific examples of plant modifications involving insertion or altered expression of regulatory proteins are discussed as illustrative case studies supporting the conclusion that the current comparative safety assessment process is appropriate for these types of biotechnology-developed crops.

A 28-day oral toxicity evaluation of small interfering RNAs and a long double-stranded RNA targeting vacuolar ATPase in mice.

New biotechnology-derived crop traits have been developed utilizing the natural process of RNA interference (RNAi). However, plant-produced double stranded RNAs (dsRNAs) are not known to present a hazard to mammals because numerous biological barriers limit uptake and potential for activity. To evaluate this experimentally, dsRNA sequences matching the mouse vATPase gene (an established target for control of corn rootworms) were evaluated in a 28-day toxicity study with mice. Test groups were orally gavaged with escalating doses of either a pool of four 21-mer vATPase small interfering RNAs (siRNAs) or a 218-base pair vATPase dsRNA. There were no treatment-related effects on body weight, food consumption, clinical observations, clinical chemistry, hematology, gross pathology, or histopathology endpoints. The highest dose levels tested were considered to be the no observed adverse effect levels (NOAELs) for the 21-mer siRNAs (48 mg/kg/day) and the 218 bp dsRNA (64 mg/kg/day). As an additional exploratory endpoint, vATPase gene expression, was evaluated in selected gastrointestinal tract and systemic tissues. The results of this assay did not indicate treatment-related suppression of vATPase. The results of this study indicate that orally ingested dsRNAs, even those targeting a gene in the test species, do not produce adverse health effects in mammals.

Computational sequence analysis of predicted long dsRNA transcriptomes of major crops reveals sequence complementarity with human genes

Long double-stranded RNAs (long dsRNAs) are precursors for the effector molecules of sequence-specific RNA-based gene silencing in eukaryotes. Plant cells can contain numerous endogenous long dsRNAs. This study demonstrates that such endogenous long dsRNAs in plants have sequence complementarity to human genes. Many of these complementary long dsRNAs have perfect sequence complementarity of at least 21 nucleotides to human genes; enough complementarity to potentially trigger gene silencing in targeted human cells if delivered in functional form. However, the number and diversity of long dsRNA molecules in plant tissue from crops such as lettuce, tomato, corn, soy and rice with complementarity to human genes that have a long history of safe consumption supports a conclusion that long dsRNAs do not present a significant dietary risk.

RNAi technologies in agricultural biotechnology: The Toxicology Forum 40th Annual Summer Meeting.

During the 40th Annual Meeting of The Toxicology Forum, the current and potential future science, regulations, and politics of agricultural biotechnology were presented and discussed. The meeting session described herein focused on the technology of RNA interference (RNAi) in agriculture. The general process by which RNAi works, currently registered RNAi-based plant traits, example RNAi-based traits in development, potential use of double stranded RNA (dsRNA) as topically applied pesticide active ingredients, research related to the safety of RNAi, biological barriers to ingested dsRNA, recent regulatory RNAi science reviews, and regulatory considerations related to the use of RNAi in agriculture were discussed. Participants generally agreed that the current regulatory framework is robust and appropriate for evaluating the safety of RNAi employed in agricultural biotechnology and were also supportive of the use of RNAi to develop improved crop traits. However, as with any emerging technology, the potential range of future products, potential future regulatory frameworks, and public acceptance of the technology will continue to evolve. As such, continuing dialogue was encouraged to promote education of consumers and science-based regulations.

Improving insect control protein activity for GM crops: A case study demonstrating that increased target insect potency can be achieved without impacting mammalian safety

&NA; Many insect‐protected crops express insecticidal crystal (Cry) proteins derived from the soil bacterium Bacillus thuringiensis (Bt), including both naturally‐occurring Cry proteins and chimeric Cry proteins created through biotechnology. The Cry51Aa2 protein is a naturally‐occurring Cry protein that was modified to increase its potency and expand its insect activity spectrum through amino acid sequence changes. The improved Cry51Aa2 variant, Cry51Aa2.834_16, and other developmental variants belong to the ETX_MTX2 family of proteins but share a low level of sequence similarity to other members of this family. This similarity is largely localized to the pore‐forming and oligomerization protein domains, while sequence divergence is observed within the head domain that confers receptor binding specificity. The intact Cry51Aa2.834_16 protein was heat labile at temperatures ≥55 °C, and was rapidly degraded after exposure to the gastrointestinal protease pepsin. No acute oral toxicity was observed in mice for three protein variants of Cry51Aa2, including Cry51Aa2.834_16, at doses exceeding 1000 mg/kg body weight. The weight‐of‐evidence therefore supports the conclusion of safety for Cry51Aa2.834_16 and demonstrates that amino acid sequence modifications can be used to substantially increase insecticidal activity of a protein without an increased hazard to mammals. HighlightsCry51Aa2.834_16 protein confers hemipteran and thysanopteran pest control in cotton.This protein was modified to selectively enhance insect control activity/spectrum.Cry51Aa2.834_16 was readily digested by pepsin and heat labile at ≥ 55 °C.Cry51Aa2.834_16 was not acutely toxic to mammals.The weight of the evidence supports food and feed safety of Cry51Aa2.834_16.