Mitochondrial DNA heteroplasmy in disease and targeted nuclease-based therapeutic approaches

Abstract

Mitochondrial DNA (mtDNA) encodes a subset of the genes which are responsible for oxidative phosphorylation. Pathogenic mutations in the human mtDNA are often heteroplasmic, where wild-type mtDNA species co-exist with the pathogenic mtDNA and a bioenergetic defect is only seen when the pathogenic mtDNA percentage surpasses a threshold for biochemical manifestations. mtDNA segregation during germline development can explain some of the extreme variation in heteroplasmy from one generation to the next. Patients with high heteroplasmy for deleterious mtDNA species will likely suffer from bona-fide mitochondrial diseases, which currently have no cure. Shifting mtDNA heteroplasmy toward the wild-type mtDNA species could provide a therapeutic option to patients. Mitochondrially targeted engineered nucleases, such as mitoTALENs and mitoZFNs, have been used in vitro in human cells harboring pathogenic patient-derived mtDNA mutations and more recently in vivo in a mouse model of a pathogenic mtDNA point mutation. These gene therapy tools for shifting mtDNA heteroplasmy can also be used in conjunction with other therapies aimed at eliminating and/or preventing the transfer of pathogenic mtDNA from mother to child.

Keywords: gene editing; heteroplasmy; mitochondrial DNA.

Figure 1. Mechanisms of mtDNA depletion during genetic bottleneck resulting in daughter cells with varying heteroplasmy

During the genetic bottleneck that occurs during germline development, there is a reduction in mtDNA levels in the fertilized oocyte and the resulting daughter cells have a wide range of heteroplasmy levels. Some possible mechanisms of the reduction in mtDNA that can explain the bottleneck during germline development include (A) the passive but marked reduction in mtDNA levels during each cell division in early development, followed by stochastic segregation of mtDNA into daughter cells; (B) mtDNA packaged into homoplasmic clusters which are reduced in discrete segregating units during each cell division; and (C) focal mtDNA replication where only a selected population of mtDNA molecules are replicated. All of these possible mechanisms would result in daughter cells with varying heteroplasmy.

Figure 2. Mechanism of mtDNA heteroplasmy shift following DNA editing enzyme‐mediated DSB

Targeting pathogenic mtDNA for degradation is one method of shifting mtDNA heteroplasmy below the biochemical threshold. (A) Staring with a cell with high levels of heteroplasmy, mitoREs, mitoTALENs, mitoTEV‐TALEs, and mitoZFNs can be used to selectively or preferentially cleave the pathogenic mtDNA molecules, resulting in a transient depletion of total mtDNA levels. Mechanisms associated with copy number control will return mtDNA to pre‐therapeutic levels but with a lower load of the pathogenic mtDNA. (B) Architecture of the DNA recognition elements and DNA editing enzymes used to shift mtDNA heteroplasmy. mitoREs are enzymes that bind to and cleave mtDNA at a specific, but short recognition sequence. mitoTALENs are comprised of two monomers, each with a DNA recognition element and one FokI monomer, when two FokI monomers are sufficiently close together they will dimerize and cleave the mtDNA. mitoTEV‐TALEs are comprised of a single monomer, with a DNA recognition element and the I‐TevI nuclease which can cleave mtDNA at a CNNNG sequence. mitoZFNs are similar to mitoTALENs in architecture, where there are two DNA recognition elements and two FokI nucleases that need to dimerize to cleave the mtDNA sequence. The difference between the DNA recognition elements in mitoTALENs/mitoTEV‐TALEs and mitoZFNs is the number of nucleotides recognized by each element (1 versus 3 nucleotides).