"These findings constitute proof of principle that mtDNA heteroplasmy correction using programmable nucleases could provide a therapeutic route for heteroplasmic mitochondrial diseases of diverse genetic origin," the authors wrote on September 24th in Nature Medicine (https://www.nature.com/articles/s41591-018-0165-9). Because mammalian mitochondria lack efficient DNA double-strand break repair pathways, the selective introduction of double-strand breaks into mutant mtDNA leads to rapid degradation of these molecules by components of the mtDNA replisome.
And, as co-author and Sangamo Therapeutics researcher Edward Rebar noted in a statement, CRISPR editing of mtDNA is currently too difficult because there are limited CRISPR editing sites on the mitochondrial genome and there is no established mechanism for importing guide RNAs into the organelle.
In previous work, however, this team described methods for the delivery of zinc-finger proteins (ZFPs) to mitochondria in cultured cells and the assembly and function of efficient mtZFN architectures that are capable of producing large heteroplasmic shifts that result in phenotype rescue of patient-derived cell cultures.
Using the m.5024C>T tRNAAla mouse model, which faithfully recapitulates key molecular features of mitochondrial disorders in cardiac tissue, the researchers set out to generate pairs of ZFPs with singlenucleotide- binding specificity for m.5024C>T. They generated a library of 24 unique ZFPs targeting the m.5024C>T site and conducted a series of screening experiments in order to select a pair for in vivo experimentation.
"Measurements of mtDNA heteroplasmy over time in cardiac tissue demonstrate significant increases in heteroplasmy-shifting activity in the latest post-treatment time points. Despite the presence of two regions with significant homology to the mtDNA target site in the nuclear genome, no evidence for offtarget effects exerted by mtZFNs could be detected at these sites," the authors wrote.
They also found a significant increase in mt-tRNAAla steady-state levels proportional to the
heteroplasmy shifts detected in the mice, as well as an altered metabolic signature in mtZFN-treated mice compared with controls, demonstrating significantly increased pyruvate levels and significantly decreased lactate levels. This suggested an improved mitochondrial respiration in the treated mice.
"Our previous reports on the use of mtZFN technology have demonstrated that these programmable nucleases can target multiple genetic lesions, producing phenotypically relevant shifts of mtDNA heteroplasmy in cellular models of mitochondrial dysfunction," the authors concluded. "Here, we have further demonstrated the flexibility and future potential of mtZFN technology by targeting another heteroplasmic mutation in mouse mtDNA."