Therapeutic avenues Louise Hyslop

1. Reproductive technologies to prevent transmission of mitochondrial DNA disease 2. Clinical trials and potential therapies for nuclear mitochondrial diseases

Mitochondria contain own DNA (mtDNA) Multiple copies of mtDNA in each cell mtDNA can have mutations Mitochondria contain own DNA (mtDNA) Multiple copies of mtDNA in each cell mtDNA can have mutations Mitochondria

MtDNA copy number (n=19) mtDNA: inherited only from our mothers Human eggs contain abundant stock of mtDNA

Mutations can be present in all, or just some copies of mtDNA Severity of disease is determined by the ratio of mutated to non-mutated mitochondrial DNA mtDNA mutations 100% mutation 0% mutation mix of mutated and normal mtDNA HomoplasmyHeteroplasmy

Mutation Load Mother Reproductive consequences Son 78% Died aged 7 years Mild clinical symptoms

Mutation Load Mother 38% Reproductive consequences Daughter 0% Son 78% Died aged 7 years Mild clinical symptoms

Mutation load Wide variation in mtDNA mutation loads between eggs and embryos 0% 78%

Summary of cases at Newcastle Fertility Centre xxxx Case C xxx x x Case D x Case B x x x x Case A x x x x x x x x Case E % mutation load

What can we offer in cases where all embryos have a high mutation load?

Mutation load Can we uncouple the inheritance of nuclear and mtDNA? Transplantation of the nuclear DNA mutant mitochondriawild-type mitochondria Are there alternative strategies? Not feasible to replace the mitochondria

Proven to be compatible with development in mice (McGrath and Solter, 1983; Meirelles & Smith, 1997) Proven to prevent transmission of a mitochondrial DNA deletion in mice (Sato et al, PNAS, 2005) Proven to be compatible with development in mice (McGrath and Solter, 1983; Meirelles & Smith, 1997) Proven to prevent transmission of a mitochondrial DNA deletion in mice (Sato et al, PNAS, 2005) Pronuclear transfer Fertilised egg Pronuclei

Donor egg Egg from affected woman mutant mitochondriawild-type mitochondria Pronuclear transfer strategy

Is it technically feasible in human fertilised eggs? Can reconstituted fertilised eggs develop? Can we minimise the level of mtDNA carryover? Is it technically feasible in human fertilised eggs? Can reconstituted fertilised eggs develop? Can we minimise the level of mtDNA carryover? In the lab: Pronuclear transfer

Craven et al, 2010, Nature, vol. 465 Embryo development: (abnormally fertilised eggs) Unmanipulated controls: 17% Pronuclear transfer: 8% Monitor embryo development

Optimisation of the procedure to minimise carry over of mtDNA Optimisation of the procedure to minimise carry over of mtDNA Before <2% Craven et al, 2010, Nature, ± 7.6% mtDNA carry-over

Donor egg Egg from affected woman mutant mitochondriawild-type mitochondria Conclusion: Pronuclear transfer is a feasible option for reducing the risk of transmission of mutated mtDNA

Effects of pronuclear transfer on embryo development –Can reconstituted embryos develop with high efficiency? –Are these reconstituted embryos normal? Are the manipulations harmful to embryos? Ongoing work: Testing Safety and Efficacy

Public consultation Debate and votes in Houses of Parliament on regulations Detailed regulations agreed and adopted – Oct 2015 Examination of the safety and efficacy data Application for licence to use new technique in clinical treatment Legal and regulatory landscape

Mitochondrial Genetics Doug Turnbull Lyndsey Craven Helen Tuppen Mitochondrial Genetics Doug Turnbull Lyndsey Craven Helen Tuppen Egg Donation Programme Alison Murdoch Meena Choudhary Maria Nesbitt Kayleigh Lennox Egg Donation Programme Alison Murdoch Meena Choudhary Maria Nesbitt Kayleigh Lennox Reproductive Biology Mary Herbert Louise Hyslop Laura Irving Jessica Richardson Dimitri Kalleas Reproductive Biology Mary Herbert Louise Hyslop Laura Irving Jessica Richardson Dimitri Kalleas Legal James Lawford Davies Legal James Lawford Davies PGD Programme Rob Taylor Bobby McFarlane Jane Stewart Charlotte Alston Sam Byerley PGD Programme Rob Taylor Bobby McFarlane Jane Stewart Charlotte Alston Sam Byerley Acknowledgements Egg donors and patients