Identification of scaffold proteins for improved endogenous engineering of extracellular vesicles

Extracellular vesicles (EVs) are gaining ground as next-generation drug delivery modalities. Genetic fusion of the protein of interest to a scaffold protein with high EV-sorting ability represents a robust cargo loading strategy. To address the paucity of such scaffold proteins, Karolinska Institutet researchers leverage a simple and reliable assay that can distinguish intravesicular cargo proteins from surface- as well as non-vesicular proteins and compare the EV-sorting potential of 244 candidate proteins. The researchers identify 24 proteins with conserved EV-sorting abilities across five types of producer cells. TSPAN2 and TSPAN3 emerge as lead candidates and outperform the well-studied CD63 scaffold. Importantly, these engineered EVs show promise as delivery vehicles in cell cultures and mice as demonstrated by efficient transfer of luminal cargo proteins as well as surface display of different functional entities. The discovery of these scaffolds provides a platform for EV-based engineering.

A bioluminescence screening protocol for quantification of luminal cargo proteins in EVs

a Selection criteria and overview of EV-sorting protein candidates. The red solid lines indicate the 25%, 50% and 75% percentile values. b SEC elution profiles of conditioned media from HEK-293T cells expressing Tluc or CD63-Tluc. Tluc activity in each fraction was quantified directly (group PBS) or after membrane lysis (group Triton) and normalized to the fraction with the highest signal. EVs and soluble proteins were recovered in fractions 0-3 and 4–12, respectively. c Scheme of differentiating Tluc forms in conditioned media. d Percentage of intravesicular Tluc for CD63-Tluc using fractionated and unfractionated media. Results are shown as the mean ± standard deviation of three biological replicates. Two-sided Student’s t test (P > 0.9999). ns: not significant. e Outline of the screening procedure and data analyses. HEK-293T cells were grown in 96-well microplates and co-transfected with Tluc fusion plasmid and Nluc plasmid. Cell cultures were centrifuged and Tluc activity was measured in the cell pellet and conditioned media. Nluc activity was only quantified in the conditioned media. c, e Created with BioRender.com. Source data are provided as a Source Data file. SEC size exclusion chromatography.