複製 Mitalipov

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MITOCHONDRIAL GENOME REPLACEMENT IN UNFERTILIZED OOCYTES FOR TREATMENT OF INHERITED MTDNA DISEASE Shoukhrat Mitalipov 1

Transcript of 複製 Mitalipov

Page 1: 複製  Mitalipov

MITOCHONDRIAL GENOME REPLACEMENT IN

UNFERTILIZED OOCYTES

FOR 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 disorder

s by replacement of mutated genes in eggs4

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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 gene

ration

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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

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Mito & Tracker 8

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Cryopreservation of oocytes before ST

Tachibana et al., Nature, 2013 9

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Organ Female #1 (%) Female #2 (%)

Cerebrum ND 0.19

Cerebellum ND ND

Heart ND 0.48

Lung ND ND

Blood ND 0.13

Stomach ND ND

Small intestine ND ND

Colon ND ND

Liver ND ND

Pancreas ND ND

Adrenal gland ND 0.2

Thyroid ND ND

Kidney-right ND ND

Kidney-left ND ND

Bladder ND ND

Uterus ND ND

Spleen ND ND

Thymus ND ND

Skin ND ND

Skeletal Muscle ND ND

ND, not detectable.

Undetectable or low mtDNA carryover in tissues and organs of

ST monkeys

Lee et al., Cell Reports, 201210

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mtDNA carryover in oocytes of ST monkeys

Individual oocytes Caryover mtDNA (%)

Female 1 Female 2

1 ND ND

2 ND ND

3 ND ND

4 0.19 ND

5 0.45 ND

6 0.45 ND

7 0.53 ND

8 1.04 ND

9 1.17 0.46

10 1.46 5.26

11 2.72 5.53

12 14.15 16.24

Lee et al., Cell Reports, 2012 11

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Normal growth and development of monkey offspring

following mtDNA replacement

Tachibana et al., Nature 201312

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• 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

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Fertilization outcomes in human zygotes following mtDNA

replacement

Tachibana et al., Nature 201315

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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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