An extracellular vesicle delivery platform based on the PTTG1IP protein

• Published in: Extracellular Vesicle (Online) 2024-12, Vol.4, p.None-None, Article 100054
• Main Authors: Martin Perez, Carla, Liang, Xiuming, Gupta, Dhanu, Haughton, Emily R., Conceição, Mariana, Mäger, Imre, EL Andaloussi, Samir, Wood, Matthew J.A., Roberts, Thomas C.
• Format: Article
• Language: English
• Subjects: CRISPR-Cas9; Exosome; Extracellular vesicle; PTTG1IP
• ISSN: 2773-0417; 2773-0417
• DOI: 10.1016/j.vesic.2024.100054

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.