Mitochondrial augmentation therapy: Preclinical Proof of Principle that Isolated Mitochondria Can Enrich Recipient Cells In Vitro

Roughly 2 billion years ago, the bacterial predecessor of mitochondria entered a symbiosis to create what we now know as eukaryotic cells. Over 40 years ago, it was first demonstrated that cells have an inherent capacity to be enriched by isolated mitochondria in culture when placed under selective pressure. Minovia is developing mitochondrial augmentation therapy, a method by which hematopoietic stem cells derived from patients with mitochondrial disease are enriched with healthy mitochondria ex vivo before reinfusion back to the patient bloodstream.

We have demonstrated that isolated mitochondria can enter cells, fuse with the endogenous mitochondrial network, and maintain functionality. Two methodologies were used to demonstrate the capacity of isolated mitochondria to enter cells and fuse with the endogenous mitochondrial network. Firstly, cells with GFP-labeled mitochondria were enriched with isolated mitochondria expressing RFP and imaged by confocal microscopy. Secondly, a split luciferase-split Venus construct was utilized to demonstrate fusion of exogenous and endogenous mitochondria after augmentation. The maintenance of functionality of internalized mitochondria was demonstrated using Rho0 cells, whose viability and proliferative capacity on medium lacking pyruvate and uridine was demonstrated after mitochondrial augmentation. Finally, when non-steroidogenic fibroblasts were augmented with placenta-derived mitochondria, the presence of internalized P450scc (cyp11a1), a placenta-specific steroidogenic enzyme, was noted, and cells transiently secreted progesterone, demonstrating functionality of internalized mitochondria. Taken together, these data support the propensity of cells to be enriched with wild type mitochondria, which can fuse with the endogenous mitochondrial network and augment mitochondrial function.