1. MITOCHONDRIAL GENOME REPLACEMENT INUNFERTILIZED OOCYTESFOR TREATMENT OF INHERITED MTDNA DISEASEShoukhrat Mitalipov1

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, heartdiseases, diabetes, cancer Inherited - neuropathy, encephalopathy, cardiomyopathy, myopathy,diabetes, metabolic syndromes Up to 4,000 children are born in the United States every year with inheritedmtDNA syndromes2 3. Complex nature of mtDNA genetics and inheritanceLebers hereditary optic neuropathy (LHON)44% 25%2% 85% 15% 52%0% 2%IIIIIIIV3 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 disorders by replacement of mutated genes in eggs4 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 tolater generations Prevents the need for repeated therapy generation after generation5 6. mtDNA replacement in oocytes Feasibility and efficacy of MII spindle-chromosomecomplex transfer (ST) Developmental Potential Mutated mtDNA carryover Nuclear/Mitochondrial genome compatibility?6 7. Mitochondrial gene replacement in oocytesSpindle imagingSeparated chromosomes (nuclear DNA) andmitochondrial DNADistribution of mitochondriain mature oocytesSpindle removal 7 8. Mito & Tracker 8 9. Cryopreservation of oocytes before STTachibana et al., Nature, 2013 9 10. Undetectable or low mtDNA carryover in tissues and organs ofST monkeysOrgan Female #1 (%) Female #2 (%)Cerebrum ND 0.19Cerebellum ND NDHeart ND 0.48Lung ND NDBlood ND 0.13Stomach ND NDSmall intestine ND NDColon ND NDLiver ND NDPancreas ND NDAdrenal gland ND 0.2Thyroid ND NDKidney-right ND NDKidney-left ND NDBladder ND NDUterus ND NDSpleen ND NDThymus ND NDSkin ND NDSkeletal Muscle ND NDND, not detectable.Lee et al., Cell Reports, 2012 10 11. mtDNA carryover in oocytes of ST monkeysIndividual oocytesCaryover mtDNA (%)Female 1 Female 21 ND ND2 ND ND3 ND ND4 0.19 ND5 0.45 ND6 0.45 ND7 0.53 ND8 1.04 ND9 1.17 0.4610 1.46 5.2611 2.72 5.5312 14.15 16.24Lee et al., Cell Reports, 2012 11 12. Normal growth and development of monkey offspringfollowing mtDNA replacementTachibana et al., Nature 201312 13. 7 egg donors A total of 106mature MII oocytesused for ST orserved as controls13 14. mtDNA replacement by Spindle Transfer (ST) in humanoocytes: efficacy, fertilization and embryo developmentTachibana et al., Nature 201314 15. Fertilization outcomes in human zygotes following mtDNAreplacement15Tachibana 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 lineswere also karyotypically abnormal (XYY or X0)16 17. Conclusions Entire cytoplasm containing mtDNA in human oocytes can beefficiently 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 producekaryotypically normal ESCs at rates similar to controls Thorough screening for abnormal fertilization is critical for selectingST embryos for transfers17 18. Clinical Trials Current efficiency allows generation of several (3-4) healthy embryosby ST suitable for embryo transfers for each cycle Recruit families carriers of early onset mtDNA diseases (at least oneaffected child, living or deceased) Recruit healthy mtDNA egg donors Conduct ST followed by PGD and/or prenatal diagnosis to ensurecomplete mtDNA replacement and chromosomal normalcy Follow up with birth and development of healthy children18