mitochondrial replacement therapy in reproductive medicine, don wolf, oregon health & science...
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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