Mäger, I. | Search bioRxiv

bioRxiv2023

An extracellular vesicle delivery platform based on the PTTG1IP protein

Extracellular vesicles (EVs) hold great promise as therapeutic delivery vehicles, although their potential is limited by a lack of efficient EV engineering strategies to enhance loading and functional delivery of therapeutic cargo. Here, we identified N-glycosylation as a putative EV- sorting feature using a novel bioinformatics analysis strategy. We exploited this finding to develop a platform for EV-mediated delivery of macromolecular cargoes based on PTTG1IP, a small N- glycosylated, single spanning transmembrane protein. We demonstrate that PTTG1IP EV enrichment is dependent on its N-glycosylation at two sites, and that PTTG1IP is a suitable scaffold for EV loading of therapeutic cargoes. To achieve functional delivery, cargoes were fused to PTTG1IP, separated by various self-cleaving sequences intended to promote cargo release from the scaffold after EV loading. In addition, the fusogenic VSVG protein was co-expressed to enhance endosomal escape. This PTTG1IP-based strategy enabled highly efficient functional delivery of Cre protein to recipient cells and mouse xenograft tumors, as well as Cas9 and Cas9/sgRNA complex delivery to reporter cells. Importantly, PTTG1IP exhibited improved protein delivery potential relative to a scaffold based on CD63 (a common EV marker). Moreover, we developed various PTTG1IP variants with improved properties, demonstrating the versatility of PTTG1IP as an EV scaffold. The EV-loading platform described in this study offers significant advantages over other strategies, such as favourable membrane topology, the potential for further engineering, and functional delivery capability, which will enable the development of improved EV-based therapeutics.

molecular biology↗

bioRxiv2020

Profiling of extracellular small RNAs highlights a strong bias towards non-vesicular secretion

Extracellular environment consists of a plethora of different molecules, including extracellular miRNA that can be secreted in association with extracellular vesicles (EVs) or soluble protein complexes (non-EVs). Yet, it is generally accepted that most of the biological activity is attributed to EV-associated miRNAs. The capability of EVs to transport cargoes has attracted much interest towards developing EVs as therapeutic short RNA carriers by using endogenous loading strategies for miRNA enrichment. Here, by overexpressing miRNA and shRNA sequences of interest in source cells and using size exclusion liquid chromatography (SEC) to separate the cellular secretome into EV and non-EV fractions, we saw that strikingly, <2% of all secreted overexpressed miRNA were found in association with EVs. To see whether the prominent non-EV miRNA secretion also holds true at the basal expression level of native miRNA transcripts, both fractions were further analysed by small RNA sequencing. This revealed a global correlation of EV and non-EV miRNA abundance to that of their parent cells and showed an enrichment only for miRNAs with a relatively low cellular expression level. Further quantification showed that similarly to the transient overexpression context, an outstanding 96.2-99.9% of total secreted miRNA at its basal level was secreted to the non-EV fraction. Yet, even though EVs contain only a fraction of secreted miRNAs, these molecules were found stable at 37{degrees}C in serum-containing environment, indicating that if sufficient miRNA loading to EVs is achieved, EVs can remain miRNA delivery-competent for a prolonged period of time. This study suggests that the passive endogenous EV loading strategy can be a relatively wasteful way of loading miRNA to EVs and active miRNA loading approaches are needed for developing advanced EV miRNA therapies in the future.

molecular biology↗