Shoukhrat Mitalipov

1. Mitochondrial genome replacement in unfertilized oocytesfor treatment of inherited mtDNA disease Shoukhrat Mitalipov

2. 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 • Acquired, age related - neurodegenerative diseases, Parkinson, heart diseases, diabetes, cancer • Inherited - neuropathy, encephalopathy, cardiomyopathy, myopathy, diabetes, metabolic syndromes • Up to 4,000 children are born in the United States every year with inherited mtDNA syndromes

3. Complex nature of mtDNA genetics and inheritance Leber’s hereditary optic neuropathy (LHON) I II 25% 44% III 15% 52% 2% 85% IV 0% 2%

4. Inherited mtDNAdiseases • mtDNA is maternally inherited - through the egg • Complex, unpredictable pattern of inheritance • These diseases are fatal or severely debilitating • No cure for mtDNA disease • Ultimate goal is to prevent transmission of mtDNA disorders by replacement of mutated genes in eggs

5. Mitochondrial Gene Replacement in Oocytes • Complete replacement of entire mtDNA • Applicable to any mtDNA mutation type • Eliminates entire spectrum of mtDNA disease • Genetic corrections will be heritable and passed on to • later generations • Prevents the need for repeated therapygeneration after generation

6. mtDNA replacement in oocytes • Feasibility and efficacy of MII spindle-chromosome complex transfer (ST) • Developmental Potential • Mutated mtDNA carryover • Nuclear/Mitochondrial genome compatibility?

7. Mitochondrial gene replacement in oocytes Distribution of mitochondria in mature oocytes Spindle imaging Separated chromosomes (nuclear DNA) and mitochondrial DNA Spindle removal

8. Mito & Tracker

9. Cryopreservation of oocytes before ST Tachibana et al., Nature, 2013

10. Undetectable or low mtDNA carryover in tissues and organs of ST monkeys Lee et al., Cell Reports, 2012

11. mtDNA carryover in oocytes of ST monkeys Lee et al., Cell Reports, 2012

12. Normal growth and development of monkey offspring following mtDNA replacement Tachibana et al., Nature 2013

13. 7 egg donors • A total of 106 mature MII oocytesused for ST or served as controls

14. mtDNA replacement by Spindle Transfer (ST) in human oocytes: efficacy, fertilization and embryo development Tachibana et al., Nature 2013

15. Fertilization outcomes in human zygotes following mtDNA replacement Tachibana et al., Nature 2013

16. 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 (17%) was triploid • 9 ESC lines from 16 control blastocysts (56%), 2 cell lines were also karyotypically abnormal (XYY or X0)

17. Conclusions • Entire cytoplasm containing mtDNAin human oocytes can be efficiently replaced by ST • Use of mt genome from donor egg (not recombinant) • Applicable to any mtDNA mutation type • ST is feasible with cryopreserved eggs • A portion of manipulated oocytes displayed abnormal fertilization • Normally fertilized zygotes develop to blastocysts and produce karyotypically normal ESCs at rates similar to controls • Thorough screening for abnormal fertilization is critical for selecting ST embryos for transfers

18. Clinical Trials • Current efficiency allows generation of several (3-4) healthy embryos by ST 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 ST followed by PGD and/or prenatal diagnosis to ensure complete mtDNA replacement and chromosomal normalcy • Follow up with birth and development of healthy children