An extracellular vesicle delivery platform based on the PTTG1IP protein.
Martin Perez C., Liang X., Gupta D., Haughton ER., Conceição M., Mäger I., El Andaloussi S., Wood MJA., Roberts TC.
Extracellular vesicles (EVs) are promising therapeutic delivery vehicles, although their potential is limited by a lack of efficient engineering strategies to enhance loading and functional cargo delivery. Using an in-house bioinformatics analysis, we identified N-glycosylation as a putative EV-sorting feature. PTTG1IP (a small, N-glycosylated, single-spanning transmembrane protein) was found to be a suitable scaffold for EV loading of therapeutic cargoes, with loading dependent on its N-glycosylation at two arginine residues. Chimeric proteins consisting of PTTG1IP fused with various cargo proteins, and separated by self-cleaving sequences (to promote cargo release), were shown to enable highly efficient functional delivery of Cre protein to recipient cell cultures and mouse xenograft tumors, and delivery of Cas9-sgRNA complexes to recipient reporter cells. The favorable membrane topology of PTTG1IP enabled facile engineering of further variants with improved properties, highlighting its versatility and potential as a platform for EV-based therapeutics.