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

MitochondriaMitochondria

MtD

NA

co

py

nu

mb

er

(n=19)

mtDNA: inherited only from our mothersmtDNA: 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 mutationsmtDNA mutations

100% mutation

0% mutation

mix of mutated and normal mtDNA

Homoplasmy Heteroplasmy

MutationLoad

Mother

Reproductive consequencesReproductive consequences

Son78%

Died aged 7 years

Mild clinical

symptoms

MutationLoad

Mother 38%

Reproductive consequencesReproductive consequences

Daughter 0%

Son78%

Died aged 7 years

Mild clinical

symptoms

Mutation load

Mutation load

Wide variation in mtDNA mutation loads between eggs and embryosWide variation in mtDNA mutation loads between eggs and embryos

0% 78%

Summary of cases at Newcastle Fertility CentreSummary of cases at Newcastle Fertility Centre

xx xx Case C

xx xx

x Case D

x

Case B

xxxx

Case A

xxxxx

x20

40

60

80

100

xx

Case E

% m

utat

ion

load

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

Mutation loadMutation load

Can we uncouple the inheritance of

nuclear and mtDNA?

Can we uncouple the inheritance of

nuclear and mtDNA?

Transplantation of the nuclear DNA

Transplantation of the nuclear DNA

mutant mitochondriawild-type mitochondria

Are there alternative strategies?Are there alternative strategies?

Not feasible to replace the

mitochondria

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)

Pronuclear transferPronuclear transfer

Fertilised egg

Pronuclei

Donor egg

Egg from affected woman

mutant mitochondriawild-type mitochondria

Pronuclear transfer strategyPronuclear 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 transferIn the lab: Pronuclear transfer

Craven et al, 2010, Nature, vol. 465

Embryo development:(abnormally fertilised eggs)

•Unmanipulated controls: 17%•Pronuclear transfer: 8%

Monitor embryo developmentMonitor embryo development

Optimisation of the procedure to minimise

carry over of mtDNA

Optimisation of the procedure to minimise

carry over of mtDNA

BeforeBefore

<2%<2%Craven et al, 2010, Nature, 465

8.1± 7.6%8.1± 7.6%mtDNA carry-overmtDNA 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

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 EfficacyOngoing 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 landscapeLegal and regulatory landscape

Mitochondrial GeneticsDoug TurnbullLyndsey CravenHelen Tuppen

Egg Donation ProgrammeAlison MurdochMeena ChoudharyMaria NesbittKayleigh Lennox

Reproductive Biology Mary HerbertLouise HyslopLaura Irving Jessica RichardsonDimitri Kalleas

LegalJames Lawford Davies

PGD ProgrammeRob TaylorBobby McFarlaneJane StewartCharlotte AlstonSam Byerley

AcknowledgementsAcknowledgements

Egg donors and patients