Predicting Recombinant mRNA Loading into Extracellular Vesicles: Insights from CD81 Fusion Constructs

Extracellular vesicles (EVs) are increasingly explored as vehicles for intercellular communication and the delivery of functional molecules, such as RNA. Recent studies have identified transcript-intrinsic features that influence EV-RNA sorting, including sequence length/complexity and coding probability. However, predicting the enrichment of coding transcripts into EVs remains exploratory. Using the workflow we previously described for the characterization of CD81 fusion constructs, we measured the vesicular distribution of recombinant transcripts transiently expressed in HEK293T cells, yielding protein cargo that mirrored the intracellular abundance. We included the CD81Δ and E7 constructs to obtain insights into the potential correlation between transcript length and EV distribution. We observed that EV-RNA levels do not scale proportionally with intracellular abundance, unlike the corresponding protein cargo. We consider cumulative RNA structure, sub-cellular dynamics, post-transcriptional modifications, and RNA-binding protein interactions as necessary factors that may dictate mRNA recruitment into EVs independent of transcript length, possibly inspiring new trajectories to maximize EV-RNA loading strategies.