Modifying the Mitochondrial Genome

Abstract

Human mitochondria produce ATP and metabolites to support development and maintain cellular homeostasis. Mitochondria harbor multiple copies of a maternally inherited, non-nuclear genome (mtDNA) that encodes for 13 subunit proteins of the respiratory chain. Mutations in mtDNA occur mainly in the 24 non-coding genes, with specific mutations implicated in early death, neuromuscular and neurodegenerative diseases, cancer, and diabetes. A significant barrier to new insights in mitochondrial biology and clinical applications for mtDNA disorders is our general inability to manipulate the mtDNA sequence. Microinjection, cytoplasmic fusion, nucleic acid import strategies, targeted endonucleases, and newer approaches, which include the transfer of genomic DNA, somatic cell reprogramming, and a photothermal nanoblade, attempt to change the mtDNA sequence in target cells with varying efficiencies and limitations. Here, we discuss the current state of manipulating mammalian mtDNA and provide an outlook for mitochondrial reverse genetics, which could further enable mitochondrial research and therapies for mtDNA diseases.

Figure 1

Transferring Mitochondria for multiple applications. Mitochondria are double-membrane bound organelles that contain their own genome (mtDNA), which is organized into nucleoprotein structures, called nucleoids, attached to the inner membrane facing the mitochondrial matrix. Transfer of exogenous mitochondria into cells that contain or lack (ρ0 cells) mtDNA could improve our understanding of ETC function, metabolism, and the interaction between the mitochondrial and nuclear genomes. Mitochondrial transfer also may hold potential for treating diseases of dysfunctional mitochondria caused by mutations in mtDNA.

Figure 2

Current methodologies for manipulating mtDNA in mammalian cells. Targeted degradation of endogenous mtDNA to shift heteroplasmy ratios can be performed with mitoTALENs or mitoZFNs. The mtDNA heteroplasmy ratio can also be shifted through a ‘bottleneck’ during the reprogramming of somatic cells to induced pluripotent stem cells (iPSCs). Adeno-associated virus (AAV) transduction can deliver up to ~5 kbp DNA into mitochondria that does not integrate into the mitochondrial genome. RNA and protein import can potentially compensate for dysfunctional mtDNA gene products. Whole mitochondria transfer technologies focus on delivering either isolated mitochondria (co-culture, microinjection, or photothermal nanoblade) or mitochondria from a donor cell (mitocytoplast, cytoplasmic fusion) to a recipient cell. Importantly, none of these methods can generate novel mtDNA sequences. To generate non-native mtDNA sequences, repair cells with mtDNA disorders, or establish cell lines with unique mtDNA mutations for basic studies and disease modeling, new methods are needed to insert, delete, or substitute sequences into existing mtDNA (‘reverse genetics’).