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

TLDR: 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.

Abstract: 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