Strategies for small RNA loading into extracellular vesicles

Small RNAs are an exciting class of therapeutics with significant untapped therapeutic potential, due to their ability to affect cell behavior at the RNA level. However, delivery of RNA is a challenge due to its size and labile nature. Extracellular vesicles (EVs) are promising as delivery vehicles due to their natural role as physiological intercellular microRNA transporters, and research has shown that EVs have significant advantages compared to competing technologies such as lipid nanoparticles. Specifically, EVs more readily transport through biological barriers, deliver RNA more efficiently, and are less immunogenic. However, intrinsic microRNA content in EVs is low and thus active small RNA loading strategies are needed to enable therapeutic use. Consequently, a variety of small RNA loading methods for EVs have been developed. These include endogenous and exogenous approaches. Exogenous approaches, in which EVs are loaded directly, have been shown to enable loading of hundreds to thousands of small RNAs per EV, but they are not readily amenable to scalable production processes. Endogenous approaches, in which EVs are loaded by upstream manipulation of the producer cell, are compatible with large scale EV production, but loading by these approaches is inconsistent and has scarcely been quantitatively analyzed. The work in this dissertation is focused on enabling small RNA therapeutics via EV delivery. The lack of an ideal small RNA loading approach for EVs is addressed by tackling important issues of both endogenous and exogenous loading. First, the loading capacity of several common endogenous loading methods was optimized and quantitatively analyzed. Additionally, new approaches to endogenous small RNA loading involving genetic manipulation of the RNA structure and the microRNA cellular processing pathway were developed and evaluated. Finally, exogenous loading via sonication was applied to enable delivery of a novel microRNA combination that was identified via a rational selection process. This combination of miR-146a, miR-155, and miR-223 was found to have potentially synergistic anti-inflammatory activity, and EV-mediated delivery of the combination opens the possibility for therapeutic application in inflammatory diseases and conditions such as sepsis. Overall, this work both improves understanding of current techniques for small RNA loading into EVs and opens new opportunities for advanced strategies, bringing EV-based small RNA therapeutics closer to clinical application

Menu labeling, calories, and nutrient density: Evidence from chain restaurants.

The Food and Drug Administration's menu labeling rule requires chain restaurants to prominently display calories, while leaving other nutritional information (e.g., fat, sodium, sugar) to the request of consumers. We use rich micronutrient data from 257 large chain brands and 24,076 menu items to examine whether calories are correlated with widely used "nutrient profile" scores that measure healthfulness based on nutrient density. We show that calories are indeed statistically significant predictors of nutrient density. However, as a substantive matter, the correlation is highly attenuated (partial R2 < 0.01). Our findings (a) suggest that the promise of calorie labeling to improve nutrient intake quality at restaurants is limited and (b) clarify the basis for transparency of nutrient composition beyond calories to promote healthy menu choices

Protein-based vehicles for biomimetic RNAi delivery

Abstract Broad translational success of RNA interference (RNAi) technology depends on the development of effective delivery approaches. To that end, researchers have developed a variety of strategies, including chemical modification of RNA, viral and non-viral transfection approaches, and incorporation with delivery vehicles such as polymer- and lipid-based nanoparticles, engineered and native proteins, extracellular vesicles (EVs), and others. Among these, EVs and protein-based vehicles stand out as biomimetically-inspired approaches, as both proteins (e.g. Apolipoprotein A-1, Argonaute 2, and Arc) and EVs mediate intercellular RNA transfer physiologically. Proteins specifically offer significant therapeutic potential due to their biophysical and biochemical properties as well as their ability to facilitate and tolerate manipulation; these characteristics have made proteins highly successful translational therapeutic molecules in the last two decades. This review covers engineered protein vehicles for RNAi delivery along with what is currently known about naturally-occurring extracellular RNA carriers towards uncovering design rules that will inform future engineering of protein-based vehicles

Combinatorial microRNA Loading into Extracellular Vesicles for Increased Anti-Inflammatory Efficacy

Extracellular vesicles (EVs) have emerged as promising therapeutic entities in part due to their potential to regulate multiple signaling pathways in target cells. This potential is derived from the broad array of constituent and/or cargo molecules associated with EVs. Among these, microRNAs (miRNAs) are commonly implicated as important and have been associated with a wide variety of EV-induced biological phenomena. While controlled loading of single miRNAs is a well-documented approach for enhancing EV bioactivity, loading of multiple miRNAs has not been fully leveraged to maximize the potential of EV-based therapies. Here, an established approach to extrinsic nucleic acid loading of EVs, sonication, was utilized to load multiple miRNAs in HEK293T EVs. Combinations of miRNAs were compared to single miRNAs with respect to anti-inflammatory outcomes in assays of increasing stringency, with the combination of miR-146a, miR-155, and miR-223 found to have the most potential amongst the tested groups.https://doi.org/10.3390/ncrna805007