## 6 RNAi Mammalian Safety
### Evaluation of Potential Activity or Adverse Effects of Ingested RNA  
The biological barriers described above collectively limit absorption of RNA molecules from the diet. Therefore, it is highly unlikely that ingested RNAs would have the capacity to regulate gene expression or induce adverse effects in a consuming mammalian organism. This concept stems primarily from a lack of significant RNA oral bioavailability [211; 212], rendering the internal dose, e.g., the number of available copies of a given RNA molecule at the putative site of action, insufficient for regulatory function [214; 219; 228]. This is made especially challenging as uptake of RNA molecules in mammals is low (e.g., less than one copy per cell), and up to 1000 to 10,000 RNA copies per cell may be required for a functional RNAi response [229]. Further complicating the possibility of plant RNA activity in the diet is that small RNAs in plants are tightly bound to Argonaute proteins. Bound RNAs are not thought to dissociate from these complexes or exchange into Argonaute proteins in the consuming organism [228]. These principles collectively provide a solid biological basis for the history of safe dietary RNA consumption described above, even when ingested sequences possess sequence complementarity to transcripts in the consuming organisms. The weight of evidence supporting the limited potential for functional activity ingested RNAs in mammals [230; 231; 232; 233] has been called into question by several peer-reviewed publications alleging the opposite. However, a series of reviews and several laboratory studies on absorption and/or activity of ingested RNAs have been published since 2012, and collectively calls into question the potential for significant uptake and bioactivity of ingested RNAs [211; 212; 213; 214; 215; 228; 234; 235; 236; 237]. For example, work challenging the concept of dietary RNA absorption/activity indicated that very low detection levels could have resulted from laboratory contamination or false-positive PCR results [238; 239; 233]. Furthermore, it is essential to ensure nutritionally balanced rodent diets when conducting rodent dietary studies. This is evidenced by one of the principal studies asserting uptake and activity of ingested RNAs in mammals [232]. Measured changes in blood cholesterol concomitant with dietary RNA detection in plasma were ultimately determined to result from dietary imbalances rather than ingested RNA activity [212]. The most comprehensive analysis of this phenomenon included an assessment of 800 human datasets, the resultsof which supported the conclusion that detection of small RNAs from exogenous sources in mammalian blood samples likely results from contamination as opposed to dietary uptake [235]. Toxicological evaluations of orally administered double-stranded RNAs have been conducted in mice to address the potential for oral activity and toxicity of these molecules. A 28-day repeated-dose oral toxicity study was conducted in mice with either a long dsRNA molecule or a pool of four 21 base pair siRNAs targeting the mouse _v-ATPase_ gene. This proof of concept study for evaluating oral activity/toxicity of RNA was conducted utilizing a known gene target for corn rootworm control when expressed in plants [55] and to then construct a long dsRNA or a pool of predicted active siRNA sequences with 100% sequence complementarity to the mouse [237]. These mouse-targeting dsRNA sequences were then administered to mice by oral gavage for 28 consecutive days at doses of \(\geq\)48 mg/kg body weight, and traditional toxicology and target gene expression endpoints were evaluated. This study did not identify oral toxicity or suppression of _v-ATPase_ gene expression in the gastrointestinal tract, kidney, liver, brain, or bone, demonstrating that biological barriers appear to preclude oral activity or toxicity of orally administered dsRNA molecules in mammals.