MITOCHONDRIAL REPLACEMENT THERAPY IN

REPRODUCTIVE MEDICINE

Don P Wolf

Center for Embryonic Cell and Gene TherapyOregon National Primate Research Center

Outline

Mitochondria 101

Preventing mtDNA transmission

Age related infertility

The future

New Scientist17Sept, 2014

Mitochondria are cytoplasmic organelles found in most

human cells

Generate most of energy – Power House

Mitochondria have their own independent genome, mtDNA

Mitochondria

John et al.2010 Human Reproduction

Mitochondrial function and mtDNA

Two genomes in one cell

nDNA

two copies

mtDNA

thousand copies

ATP

Diseases caused by mtDNA mutations

There are more than 700 known disease-associated mtDNA mutations

(mitomap.org)

- 285 tRNA/rRNA

- 266 protein coding and control region point mutations;

- 131 deletions

Inherited - neuropathy, encephalopathy, cardiomyopathy, myopathy,

diabetes, metabolic syndromes

Acquired, age related - neurodegenerative diseases, Parkinson, ALS,

heart diseases, diabetes, cancer

mtDNA patient skin fibroblasts

MELAS: 40 years old male, 3243 A>G, MT-TL1

gene, 30% heteroplasmy

mitochondrial encephalomyopathy, lactic acidosis and stroke-like

episodes, multisystem disorder with generalized tonic-clonic seizures,

recurrent headaches, anorexia with recurrent vomiting and postlingual

hearing loss

Leigh disease: 8 month old male, 8993 T>G,

ATP6 gene, homoplasmic

psychomotor retardation or regression, hypotonia, spasticity, movement

disorders (including chorea), cerebellar ataxia, and peripheral neuropathy,

hypertrophic cardiomyopathy

Preventing transmssion of inherited mtDNA

disorders

Genetic counseling - adoption of children, embryos

or gametes

Preimplantation or Prenatal Genetic Diagnosis

Preimplantation Genetic Diagnosis

healthy embryo

discard discard

embryos with mutation embryos with mutation

• PGD attempt for a family with 3243A>G MELAS mutation

• 12% mutation load embryo transfer resulted in birth of a boy

• Claim: This study represents the first successful clinical application of PGD to reduce the transgenerational risk of transmitting a mtDNA disorder

• Child’s Phenotype: Preterm birth due to preeclampsia with atretic placenta, hypoxemia, recurrent hypoglycemia, recurrent infection with projectile vomiting, febrile seizure, gastrointestinal reflux, mild developmental delay, dysmorphic features with coarse facies, behavioral disorder, repeated small strokes, multiple puntcate signal abnormalities throughout brain on MRI

• Independent mtDNA testing revealed 42-52% mtDNA mutation load in blood and urine

Fertil Steril 2012

Three-parent IVF

Germline Gene Therapy

Correction or replacement of mutated genes in germ

cells (eggs, sperm, preimplantation embryos)

Genetic corrections will be heritable and passed on to

later generations

Prevents the need for repeated somatic gene therapy

every generation

Germline gene therapy is ethically challenged

Mitochondrial gene therapy via spindle transfer (ST)

Mito & Tracker

Mitochondrial gene replacement in oocytes

Spindle imaging

Separated chromosomes (nuclear DNA) and

mitochondrial DNA

Distribution of mitochondria

in mature oocytes

Spindle removal

Tachibana et al., Nature, 2013

Tachibana et al., Nature, 2013

ESC lines from human ST and control embryos

5 ESC lines from 13 human ST blastocysts (38%)

contained normal euploid karyotypes

mtDNA carryover 1% or lower

1 ESC line from 6 abnormally fertilized ST

blastocysts was triploid

9 ESC lines from 16 control blastocysts (56%), 2 cell

lines were also karyotypically abnormal (XYY or X0)

SCNT in human oocyte

MRT Highlights

Use of mt genome from donor egg

Applicable to any mtDNA mutation type

Replacement of deficient cytoplasm in patient oocytes

Preclinical animal studies demonstrate safety and efficacy

Approved in UK for clinical trials

Current efficiency allows generation of several (3-4) healthy

embryos suitable for embryo transfers for each cycle

Recruit families –carriers of early onset mtDNA diseases (at

least one affected child, living or deceased)

Recruit healthy mtDNA egg donors

Conduct MRT followed by PGD and/or prenatal diagnosis to

ensure complete mtDNA replacement and chromosomal

normalcy

Follow up with birth and development of healthy children (3-5

years)

Clinical Trials

Advanced Maternal Age and Reproduction

• Approximately 20% of women wait until after age 35 to

begin their families

• A 30-year-old woman has a 20% chance per month to

get pregnant

• By age 40, however, this chance is 5% or less per

month

IVF Use by Age Group in the US

Total IVF patients in 2008

148,055

Center for disease control and prevention 2008 ART report

IVF Success Rates

Center for Disease Control and Prevention 2009 ART Report

Clinical Pregnancy Rate

Risk of Miscarriage with AgeA

bo

rtio

n (

%)

Center for disease control and prevention 2008 ART report

Live Birth with Own or Donor Eggs

Women’s Age

Center for Disease Control and Prevention 2008 ART Report

Mitochondrial Involvement in Oocyte Aging

• Increased mtDNA mutations in eggs

• Decreased metabolic activity in embryos

• Decreased ATP production

• Altered calcium homeostasis

The Future

• Mitochondrial replacement in monkeys and humans is compatible with normal embryonic development and ESC derivation. Next, controlled trials must be carried out

• Oocyte age related fertility trials with donor mitochondria/cytoplasts

• Germ line gene editing is coming despite concerns by the scientific community