Unravelling the effects of mitochondrial replacement therapy on mtDNA transmission

Lead Supervisor: Professor Dagan Wells

Co-supervisors: Professor Joanna Poulton, Dr Ana Victoria Lechuga-Vieco, Dr Brent Ryan

Commercial Partner: Juno Genetics

Mitochondria replacement therapy (MRT) is a translational therapy undergoing clinical trial, for (i) preventing mitochondrial DNA (mtDNA) disease and (ii) treating female infertility. Normal oocytes contain ~100,000 copies of mtDNA, which are effectively identical. Patients with mtDNA disease may harbour both mutant and normal mtDNA (heteroplasmy). 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 resulted in embryos with 99% of mtDNA derived from the donor oocyte, heteroplasmy increased to >35% in 2/6 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, potentially resulting in pathogenic levels of defective mitochondria.

It is therefore vital to understand the cause and effect of heteroplasmic shifts, to examine potential agents that can modify it, and comprehend any resulting alterations in cellular function among the donors. This study aims to address these questions:

1) In collaboration with AVLV we will perform a comprehensive metabolic immune profiling using spectral flow cytometry in peripheral blood mononuclear cells (PBMCs) donated by the offspring from DW’s clinical study, compared with age-matched and parental controls. We will address how mitophagy shapes immune cell transcriptional programs, cytokine production and inflammatory responses using single-cell RNAseq in donors with different heteroplasmy levels.

2) Using cultured cells from vertebrate models of heteroplasmy we will

2.1 Quantify mitophagy and the rate of replication of the two mtDNA strains using
• high throughput imaging (Operetta, Perkin Elmer) 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

2.2 Explore drug modulators of mitophagy identified as part of BR’s ongoing drug discovery program
This project provides training in MRC priority skills by being quantitative and aimed at industrial priorities advanced therapies, translational development and precision healthcare. 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. A method that is able to assess the effectiveness of mitophagy in targeting heteroplasmic variants 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.

Apply using course: DPhil in Women's and Reproductive Health