(684e) Extracellular Vesicles Incorporating Retrovirus-like Capsids for the Enhanced Packaging and Systemic Delivery of mRNA into Neurons

The blood–brain barrier (BBB) restricts the systemic delivery of messenger RNAs (mRNAs) into diseased neurons. Although leucocyte-derived extracellular vesicles (EVs) can cross the BBB at inflammatory sites, it is difficult to efficiently load long mRNAs into the EVs and to enhance their neuronal uptake. Addressing this, our research introduces a strategy employing extracellular vesicles (EVs) derived from leukocytes, engineered with endogenous virus-like components for the systemic delivery of mRNA into neurons.

These engineered retrotransposon Arc EVs (eraEVs) are created by genetically modifying leukocytes to express the Arc protein, which forms capsids around mRNA, further stabilized by Arc 5’-untranslated-region RNA elements, enhancing their ability to package and deliver mRNA efficiently and stably. To characterize the stability, composition, and delivery efficacy of eraEVs, we employed a variety of analytical techniques, including mass spectrometry, RT-qPCR following RNA immunoprecipitation, flow cytometry, in vivo imaging, immunohistochemistry, and confocal microscopy. We first identified the crucial role of the A5U RNA regulatory motif in establishing stable Arc EVs, which substantially enhanced their ability to encapsulate and transport cargo mRNAs. The incorporation of Arc alone increased mRNA loading into EVs, and the addition of the A5U motif further stabilized the Arc capsids, leading to improved mRNA packaging. With this combination, there was a marked increase in mRNA uptake in in vitro recipient cells. Furthermore, we demonstrated the capability of leucocyte-derived eraEVs to transport mRNA into neurons of brains exhibiting chronic pan-neuronal inflammation. Altogether, we developed an efficient and safe nanocarrier for targeted mRNA delivery.

This study presents a method for using endogenous retrovirus-like vesicles as mRNA drug-delivery systems. Apart from the enhancements in cargo loading, our system offers several additional advantages. First, produced from self-derived leucocytes, eraEVs are immunologically inert and actively enriched in inflammatory microenvironments crossing the BBB with the help of membrane molecules from donor leucocytes. Also, Arc components recruit enveloping proteins during self-assembly and subsequently promote cellular uptake of EVs by recipient neurons, based on their native functions in mediating inter-neuronal exchange of molecules. Moreover, the virus-like capsid makes eraEVs more stable than other engineered RNA-loading EVs, protecting the cargo from RNase degradation until its release is triggered. In addition to taking advantages of these unique virus-like features, eraEVs are safe. They function as transient drug carriers without the ability to replicate, infect or insert genetic information into the recipient genome. Importantly, eraEVs can be produced from most donor cells and used in a wide range of biomedical applications, based on the native targeting ability of EVs from different cell types. We show an endogenous virus-like system capable of loading and delivering mRNA in vivo, particularly into disease neurons via systemic administration.