Unravelling the effects of mitochondrial replacement therapy on mtDNA transmission
PROJECT TITLE
SUPERVISORS
Juno Genetics (Commercial partner)
DESCRIPTION OF PROJECT
Normal oocytes contain ~100,000 copies of mtDNA, which are effectively identical. Patients with mtDNA disease may harbour both mutant and normal mtDNA (heteroplasmy). Mitochondria replacement therapy (MRT) is a translational therapy undergoing clinical trial, for (i) preventing mitochondrial DNA (mtDNA) disease and (ii) treating female infertility. In MRT, maternally transmitted mitochondria are replaced with healthy mitochondria using an enucleated donor oocyte to reduce the maternally transmitted dose of mtDNA. Early embryos, derived by MRT, usually contain < 5% of maternal mitochondrial DNA.
DW generated data in the Juno diagnostic facility showing that even though MRT performed for infertility replaced all but ~2% of maternal mtDNA initially, the heteroplasmy increased to >50% in 1/6 of the offspring. This could be due to a difference in replication rates of the two mtDNA variants or by altered mtDNA quality control by mitophagy. The heteroplasmy shift could be damaging and might cause immunological alterations and premature aging, as previously reported by AVLV in experimental mouse models of heteroplasmy. It could have serious consequences for efficacy if it occurred following MRT to prevent mtDNA disease transmission.
We aim to investigate the cause and effect of this heteroplasmic shift, examine potential agents that can modify it, and comprehend any resulting alterations in cellular function among the donors.
Using cultured cells from mouse models of heteroplasmy we will;
• Quantify mitophagy and the rate of replication of the two mtDNA strains using high throughput imaging (InCell 1000) and three validated mitophagy assays, specific for different stages, validated in JP’s lab.
• Develop a quantitative assay for mitophagy quality using next generation sequencing
• Develop an assay for rate of specific mtDNA clade replication based on JPs previous pulse labelling assays
• Use inducers of mitophagy targeting different aspects of mitophagy/specific types of mitochondria, validated as part of BR’s program to identify compounds which reduce hetroplasmy.
In vivo analysis
We will conduct a thorough immunoprofiling using peripheral blood mononuclear cells (PBMCs) provided by offspring, alongside age-matched and parental controls from the DW clinical study in collaboration with AVLV. Employing cutting-edge techniques such as single-cell RNA sequencing, mitochondrial FACS, and spectral flow cytometry, we will clarify the influence of mitochondrial heterogeneity and mitophagy on immune cell transcriptional program and inflammatory responses.
The analyses that Juno genetics will make available to the student, including high depth next generation sequencing of mtDNA, will reduce the costs of the project substantially. Generating a novel pipeline which can be used to explore whether mitophagy is effective in heteroplasmic cells from mice and humans will be a useful tool for the academic community. Identifying potential treatments to reduce heteroplasmy by activating mitophagy may be useful for the children who have already been born. This study will benefit Juno Genetics by focussing on the safety implications of this innovative technique, particularly its impact on the immune responses of the offspring. Furthermore, the project offers valuable training in MRC and industrial priority skills, encompassing areas such as advanced therapies, translational development, and preventative and precision medicine.
HOW TO APPLY
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