Dr.Med. Candidate Heidelberg University Heidelberg, Germany
Abstract: Osteoarthritis (OA) treatment requires effective inflammation mitigation. Interleukin-10 (IL10), an anti-inflammatory protein, shows therapeutic potential but faces challenges in direct application due to delivery inefficiency. Mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) present a promising delivery platform but are limited by replicative senescence, low exosome yield, and inconsistent therapeutic outcomes.
Here, we achieved reversible immortalization of human umbilical MSCs using lentivirally delivered excisable hTERT and SV40T transgenes, extending proliferation capacity while preserving stem cell properties. To produce IL10-enriched MSC exosomes, we employed two strategies: (1) engineering an N-terminal peptide sequence using the XPack lentivector to target IL10 to the exosome membrane and (2) utilizing CRISPR/dCas9 activation to enhance endogenous IL10 transcription. Combining these approaches enabled the large-scale production of bioengineered MSC_IL10_EVs, which were tested in an in vitro OA model for inflammation reduction.
In vitro, MSC_IL10_EVs facilitated the polarization of CD86+ M1 macrophages into MERTK+CD206+ M2 macrophages and reduced levels of TNF-α and IL-1β, effects reversible by IL10 receptor inhibition. In vivo, MSC_IL10_EVs improved gait abnormalities in an OA rat model.
These findings demonstrate an efficient platform for producing therapeutic exosomes, enhancing OA treatment efficacy through MSC-based IL10 delivery systems.
