Development of digital PCR method to quantitate exogenous mitochondrial DNA in mitochondrially-augmented cells

Mitochondria, vital cellular organelles responsible for the production of cell energy, contain their own mitochondrial DNA (mtDNA). Mutations in the mtDNA are known to cause various diseases with severity ranging from mild symptoms to life threating syndromes. We have developed a method, termed mitochondrial augmentation, to enrich cells with exogenous mitochondria ex vivo. Mitochondrial augmentation is being tested in clinical trials with the aim of alleviating symptoms of mitochondrial diseases.

To ascertain the biodistribution and bio-persistence of exogenous mtDNA after mitochondrial augmentation, we used an in vivo mouse model in which bone marrow cells from FVB/N mice were enriched with C57BL mitochondria and reinfused to FVB/N recipient mice. We developed a digital PCR-based method to assess the abundance of exogenous (C57BL) vs endogenous (FVB/N) mtDNA based on SNPs which differentiate between their mtDNA sequences. Using DNA isolated from these two strains, we calibrated quantitation of mtDNA levels of C57BL and FVB/N mtDNA. Using this system, we demonstrated a dose-dependent increase in augmentation of murine BM cells in vitro. In vivo, persistence of C57BL-derived mtDNA was demonstrated in FVB/N mice in hematopoietic and non-hemopoietic organs for up to three months after treatment. These results suggest a biodistribution of exogenous mtDNA that could result from recruitment of BM cells to different organs. As it has previously been demonstrated that injured tissue releases SDF-1, a chemokine which recruits BM cells, it is possible that injured organs, such as in mitochondrial disease mouse models, will recruit mitochondrially augmented BM cells to even higher levels.