MITOCHONDRIAL GENOME REPLACEMENT IN UNFERTILIZED

OOCYTESFOR TREATMENT OF INHERITED MTDNA

DISEASE

Shoukhrat Mitalipov

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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

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Complex nature of mtDNA genetics and inheritance

44% 25%

2% 85% 15% 52%

0% 2%

I

II

III

IV

Leber’s hereditary optic neuropathy (LHON)

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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 eggs

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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

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mtDNA replacement in oocytes

Feasibility and efficacy of MII spindle-chromosome complex transfer (ST)

Developmental Potential

Mutated mtDNA carryover

Nuclear/Mitochondrial genome compatibility?

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Mitochondrial gene replacement in oocytes

Spindle imaging

Separated chromosomes (nuclear DNA) and mitochondrial DNA

Distribution of mitochondria in mature oocytes

Spindle removal 7

Mito & Tracker 8

Cryopreservation of oocytes before ST

Tachibana et al., Nature, 20139

Undetectable or low mtDNA carryover in tissues and organs of ST monkeys

Lee et al., Cell Reports, 201210

mtDNA carryover in oocytes of ST monkeys

Lee et al., Cell Reports, 201211

Normal growth and development of monkey offspring following mtDNA replacement

Tachibana et al., Nature 201312

• 7 egg donors

• A total of 106 mature MII oocytes used for ST or served as controls

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mtDNA replacement by Spindle Transfer (ST) in human oocytes: efficacy, fertilization and embryo development

Tachibana et al., Nature 201314

Fertilization outcomes in human zygotes following mtDNA replacement

Tachibana et al., Nature 201315

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)

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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

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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

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