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Updated: Mar 10, 2026
An In Vitro Approach to Study Mitochondrial Dysfunction: A Cybrid Model
Published on: March 9, 2022
Mitochondrial replacement in human oocytes carrying pathogenic mitochondrial DNA mutations
Eunju Kang1,2, Jun Wu3, Nuria Marti Gutierrez1,2
1Center for Embryonic Cell and Gene Therapy, Oregon Health &Science University, 3303 SW Bond Avenue, Portland, Oregon 97239, USA.
Abstract:
Maternally inherited mitochondrial (mt)DNA mutations can cause fatal or severely debilitating syndromes in children, with disease severity dependent on the specific gene mutation and the ratio of mutant to wild-type mtDNA (heteroplasmy) in each cell and tissue. Pathogenic mtDNA mutations are relatively common, with an estimated 778 affected children born each year in the United States. Mitochondrial replacement therapies or techniques (MRT) circumventing mother-to-child mtDNA disease transmission involve replacement of oocyte maternal mtDNA. Here we report MRT outcomes in several families with common mtDNA syndromes. The mother's oocytes were of normal quality and mutation levels correlated with those in existing children. Efficient replacement of oocyte mutant mtDNA was performed by spindle transfer, resulting in embryos containing >99% donor mtDNA. Donor mtDNA was stably maintained in embryonic stem cells (ES cells) derived from most embryos. However, some ES cell lines demonstrated gradual loss of donor mtDNA and reversal to the maternal haplotype. In evaluating donor-to-maternal mtDNA interactions, it seems that compatibility relates to mtDNA replication efficiency rather than to mismatch or oxidative phosphorylation dysfunction. We identify a polymorphism within the conserved sequence box II region of the D-loop as a plausible cause of preferential replication of specific mtDNA haplotypes. In addition, some haplotypes confer proliferative and growth advantages to cells. Hence, we propose a matching paradigm for selecting compatible donor mtDNA for MRT.
Insights
Mitochondrial replacement therapy (MRT) can prevent children from inheriting debilitating mitochondrial DNA (mtDNA) mutations. Donor mtDNA compatibility, influenced by replication efficiency and specific polymorphisms, is crucial for stable maintenance in embryonic stem cells.
Area of Science:
- Genetics
- Reproductive Medicine
- Cell Biology
Background:
- Maternally inherited mitochondrial DNA (mtDNA) mutations cause severe childhood syndromes, with disease severity linked to heteroplasmy levels.
- Pathogenic mtDNA mutations affect approximately 778 children annually in the US, highlighting the need for preventative strategies.
- Mitochondrial replacement therapies (MRT) aim to prevent transmission of these mutations by replacing maternal mtDNA in oocytes.
Purpose of the Study:
- To evaluate the outcomes of mitochondrial replacement therapies (MRT) in families affected by common mitochondrial DNA (mtDNA) syndromes.
- To investigate the stability and maintenance of donor mtDNA in embryonic stem cells (ES cells) following MRT.
- To identify factors influencing donor-mtDNA compatibility and propose a matching paradigm for MRT.
Main Methods:
- Oocyte spindle transfer was used to replace maternal mtDNA with donor mtDNA in embryos from families with mtDNA syndromes.
- Embryonic stem cells (ES cells) were derived from resulting embryos to assess donor mtDNA stability.
- Analysis of mtDNA interactions, replication efficiency, and genetic polymorphisms to determine compatibility factors.
Main Results:
- Spindle transfer successfully replaced maternal mtDNA, achieving >99% donor mtDNA in resulting embryos.
- Donor mtDNA was stably maintained in most derived ES cell lines, but some showed gradual loss and reversal to the maternal haplotype.
- mtDNA compatibility appears to be linked to replication efficiency and specific polymorphisms (e.g., in the D-loop's conserved sequence box II), rather than simple mismatch or oxidative phosphorylation function.
Conclusions:
- Mitochondrial replacement therapy is effective in replacing mutant maternal mtDNA, but long-term stability of donor mtDNA is critical.
- Specific mtDNA polymorphisms influence replication efficiency and confer cellular growth advantages, impacting donor-mtDNA compatibility.
- A matching paradigm based on selecting compatible donor mtDNA, considering replication efficiency and polymorphisms, is proposed for successful MRT.
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