Super mitochondria-enriched extracellular vesicles enable enhanced mitochondria transfer

Mitochondria transfer is a spontaneous process that releases functional mitochondria to damaged cells via different mechanisms including extracellular vesicle containing mitochondria (EV-Mito) to restore mitochondrial functions. However, the limited EV-Mito yield makes it challenging to supply a sufficient quantity of functional mitochondria to damaged cells, hindering their application in mitochondrial diseases. Here, we show that the release of EV-Mito from mesenchymal stem cells (MSCs) is regulated by a calcium-dependent mechanism involving CD38 and IP3R signaling (CD38/IP3R/Ca²⁺ pathway). Activating this pathway through our non-viral gene engineering approach generates super donor MSCs which produce Super-EV-Mito with a threefold increase in yield compared to Ctrl-EV-Mito from normal MSCs. Leber's hereditary optic neuropathy (LHON), a classic mitochondrial disease caused by mtDNA mutations, is used as a proof-of-concept model. Super-EV-Mito rescues mtDNA defects and alleviates LHON-associated symptoms in LHON male mice. This strategy offers a promising avenue for enhancing mitochondria transfer efficiency and advancing its clinical application in mitochondrial disorders.