## 6 RNAi Mammalian Safety
### Biological Barriers to RNA Absorption  
Owing to the ubiquitous presence of the RNAi mechanism in eukaryotic organisms, RNA molecules are ingested by vertebrates through the consumption of plants, animals, and fungi. Such RNA molecules include those with double-stranded regions that could initiate RNAi if they were to be absorbed from the diet and reach cells following consumption by the organism. As a further illustration of this point, exogenous dietary RNAs include those with sequence complementarity to vertebrate genes [195; 196; 197; 207; 208]. As vertebrates are constantly exposed to such RNA molecules, it is not surprising that there are a series of biological barriers that preclude these dietary components from serving in a regulatory capacity, and instead, they are harnessed for nutritional value. These biological barriers are reviewed in the scientific literature [198; 199; 209].  
Ingested RNAs face an acidic digestive environment in the stomach following their initial exposure to nucleases in the saliva [210]. The efficacy of the low pH and hydrolysis in the stomach for nucleic acid degradation and the removal of bases from the nucleic acid backbone (e.g., depurination) has been well described and reviewed in the literature [198; 209; 211]. Further digestion of ingested nucleic acids occurs in the small intestine due to the presence of digestive enzymes and nucleases secreted by the pancreas [209]. Due to this extensive collection of barriers, nucleic acids from the diet are extensively degraded and do not undergo substantive absorption in an intact form, as demonstrated empirically with miRNAs in rodents [211; 212], rhesus monkeys [213], and humans [214]. Thorough reviews of the safety considerations of plant expressed and externally applied RNA molecules for humans and other vertebrates have been published [198; 215].  
Biological barriers to the absorption and function of ingested nucleic acids expand beyond digestive barriers and include cellular membranes impermeable to RNAs that are both highly polar and large [198; 216]. Each successive series of membrane barriers must be crossed for a dietary RNA to move from the intestinal tract lumen, across endothelial cells, and into the bloodstream. Once in the blood, RNAs are then subjected to nucleases [217; 218; 219] and rapid renal elimination [217; 220]. To reach a putative target tissue, any RNA molecule escaping these nucleases would then have to cross through the endothelium and the epithelium within a target tissue to have the capacity to modulate tissue gene expression. Owing to the impermeability of these charged macromolecules across membrane barriers [198; 216], any RNA surviving the intestinal tract would be unlikely to reach the bloodstream or a target tissue. Furthermore, RNAs reaching the cytoplasm of a cell in the consuming organism would subsequently be subjected to sequestration into endosomes that retain a vast majority of exogenous RNA molecules [221; 222].  
This array of physical, chemical, and biological barriers has made the development of pharmaceutical RNA drugs challenging, necessitating their chemical stabilization to limit degradation and specialized delivery formulations to elude barriers to exogenous RNA molecules [209; 221; 223; 224]. Without such formulations, injection of these exogenous RNA molecules results in their rapid degradation and elimination [217; 220; 225; 221]. Oral delivery of therapeutic macromolecules represents a desirable route of administration that is even more elusive to drug developers than systemically or locally administered therapeutics due to the aforementioned biological barriers [221; 224]. These challenges are evident from literature reviews and studies demonstrating the need for specialized formulations and/or chemical modifications to facilitate limited delivery within proof-of-concept oral delivery evaluations of RNA therapeutics [209; 223; 226; 227].