1. M ITOCHONDRIAL GENOME REPLACEMENT IN UNFERTILIZED OOCYTES FOR TREATMENT OF INHERITED MT DNA DISEASE Shoukhrat Mitalipov 1

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 2

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

4. Inherited mtDNA diseases 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 dis orders by replacement of mutated genes in eggs 4

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 therapy generation after generation 5

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

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

8. Mito & Tracker 8

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

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

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

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

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

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

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

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) 16

17. Conclusions Entire cytoplasm containing mtDNA in 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 17

18. 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 Clinical Trials 18