Mitochondrial genome engineering in the murine germline using designer nuclease technology
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Mitochondria are subcellular organelles with numerous roles in metabolic and cellular pathways. Most notably, mitochondria produce ATP and energetic intermediates through oxidative phosphorylation (OXPHOS). Most of the approximately 1500 proteins needed for a fully functional mitochondrion are encoded in the nuclear genome. However, the mitochondrial genome, which is a circular, multi-copy genome of roughly 17 kb in size, also encodes for 13 polypeptide genes that form key components of the OXPHOS complexes, along with the 22 tRNA genes and 2 rRNA genes required for their expression. As with any other genetic material, mutations in mitochondrial DNA (mtDNA) can have serious consequences and can lead to mitochondrial diseases. Diseases arising from mutations within mtDNA can have effects on any tissue, although tissues with high energy demand, such as the brain, the muscles and the eyes, experience greater deleterious effects. A large proportion of diseases caused by mutations in mtDNA occur in a heteroplasmic manner, whereby wild-type and mutant mtDNA haplotypes co-exist within a cell. While generally a mutation load of greater than 60% of mutated mtDNA is required to manifest in mitochondrial disease, this is dependent on the exact mutation. In this way, the ratio of wild-type to mutant mtDNA represents the penetrance of the mitochondrial disease phenotype. Diseases arising from mtDNA mutations are often fatal and at this point are completely incurable. Over recent years, approaches have been developed to selectively eliminate mutated mtDNA, allowing for the re-population of wild-type mtDNA within a cell. This has been achieved by the application of designer nuclease technologies, where engineered DNA binding domains, consisting of either a dimeric zinc finger protein (ZFP) or a transcription activator-like effector (TALE) domain, is conjugated to a dimeric nuclease domain, producing zinc finger nucleases (ZFN) or TALE-nucleases (TALEN), respectively. By targeting these pairs of engineered nucleases adjacent to a site of interest, a DNA double-strand break (DSB) can be produced, leading to degradation of the mtDNA molecule. Using novel architecture to express and localise these engineered nucleases it has been possible to target these nucleases directly to mitochondria (mtZFN, mitoTALEN) in cell culture. When expressing a mtZFN targeting a nuclear gene, this novel architecture seems to be successful in that the majority of protein localises to mitochondria, however, it has proven insufficient to prevent mutations within the nuclear genome, demonstrating the potential risk of off-target effects caused by designer nuclease technology. The recent development of a pathogenic mtDNA disease mouse model, containing a m.5024C>T mutation on the tRNAAla gene within the mitochondrial genome has provided an in vivo model for the validation of the technique. My research includes in vitro experiments demonstrating the expression, localisation and capacity to selectively degrade mutant mtDNA molecules in m.5024C>T mouse embryonic fibroblasts with both mtZFN and mitoTALEN, leading to a shift in heteroplasmy towards a greater proportion of wild-type mtDNA. Very recently, both, the Minczuk and Moraes laboratories have independently demonstrated, that AAV-targeted delivery of designer nucleases in the tRNAAla mouse, could selectively eliminate mutated mtDNA in vivo by use of either designer nuclease technologies. My research focused on adapting this technique for use in mouse embryos. I was able to demonstrate many of the technical and biological obstacles that have to be eliminated to move closer toward in vivo expression, localisation and selective degradation of mutated mtDNA by use of either mtZFN or mitoTALEN in mouse embryos. Through extensive optimisation steps in designer nuclease synthesis, advancement in embryo culture and embryo handling techniques, revising microinjection setups and making more general experimental improvements I was able to achieve in vivo expression and an implied localisation of designer nucleases to mitochondria in the mouse embryo. Additionally, I was able to showcase a shift in mtDNA heteroplasmy and reduction mtDNA copy number after mitoTALEN injections, while maintaining normal and healthy embryo development. While this procedure remains far from being used as a potential treatment for mitochondrial diseases in humans, this work has elucidated the difficulty, hurdles and labour-intensive state of techniques that must be addressed when using this technology to specifically eliminate mitochondrial disease-causing mutations in human embryos.