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A New Finding Could Improve Efficacy and Safety of Mitochondrial Replacement Therapy

Wexler, Alyse

doi: 10.1097/01.NT.0000512101.10292.36
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Researchers learned that the pairing of mothers and donors whose mitochondrial genomes have similar non-coding sequences may be key to protecting the safety and effectiveness of mitochondrial replacement therapy, preventing residual pathogenic maternal mitochondrial DNA from repopulating the rescued embryo.

Researchers at the Oregon Health and Science University provided evidence in human cellular studies that mutated mitochondrial DNA from the egg of mothers with children affected by Leigh syndrome and MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes) could be effectively replaced with the non-pathogenic mitochondrial DNA of a donor, and the reconstructed egg could produce a healthy embryo, according to a paper in the December 8, 2016 issue of Nature.

In these cases, the problem is not with the mother's healthy nucleus and its associated genetic material, but with the maternal mitochondrial DNA (mtDNA), and the point of the transfer is to put that healthy nucleus into a donor cell, which also has healthy mitochondrial DNA.

In the current study, the researchers learned that the pairing of mothers and donors whose mitochondrial genomes have similar non-coding sequences may be key to protecting the safety and effectiveness of mitochondrial replacement therapy (MRT), preventing residual pathogenic maternal mitochondrial DNA from repopulating the rescued embryo.

“You could select an embryo with less mutations than others, hoping that this will not affect the child as much, but because the mutation is still there, the chances of having a completely healthy baby are slim,” said senior study author Shoukhrat Mitalipov, PhD, director of the Center for Embryonic Cell and Gene Therapy at OHSU. “Mitochondrial replacement therapies would allow families carrying these mutations to have another child, but with mutations corrected.”

Dr. Mitalipov had previously developed a MRT technique in macaque monkeys, removing the nucleus of a mother's egg and transferring it into a donor egg stripped of its nucleus, producing healthy twin macaques.

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In the current study, Dr. Mitalipov and colleagues collected the oocytes from four women who have children with Leigh syndrome and MELAS and donor eggs from 11 healthy women, screened to confirm they did not carry inherited pathogenic mutations in their mtDNA. Subsequent genetic testing of blood, skin fibroblasts, and urine revealed that the mitochondrial disease of one of the families was not maternally inherited, so only three families were eligible for the replacement therapy.

The researchers transferred nuclear spindles from the disease-carrying oocytes into enucleated donor eggs of healthy women, and the reconstructed oocytes were then fertilized via intracytoplasmic sperm injection.

For the most part, the results were promising — six healthy blastocyst-stage embryos were produced from carrier spindle transfers, which was comparable to controls (p>005), and most of the cell lines had less than 1 percent of the diseased mitochondria.

“When we replace mtDNA we try to do it thoroughly, but because we're dealing with about a half-million copies of mtDNA in the egg, moving it all and replacing with the donor's is not a simple task,” explained Dr. Mitalipov. “That 1 percent [maternal mtDNA] was always kind of out there, but we thought it would stay at 1 percent. And 1 percent mutation, as far as we know, does not cause problems for cells, or the muscles or other vital organs and tissues, so there would be no disease. It was always thought that you could disregard it.”

But while the majority maintained greater than 99 percent donor mtDNA at 10 weeks, four of 26 blastocyst-derived embryonic stem cell lines reverted back to the maternal mtDNA haplotype.

To try to learn more about why the reversal occurred, the researchers zeroed in on a portion of the mtDNA known as the non-coding D-loop region, which initiates replication of the entire genetic sequence. They found that two of the four embryonic stem lines that reverted back to the diseased maternal mtDNA contained guanosine additions in the region of the maternal D-loops, while the healthy donor lines carried guanosine deletions.

The phenotype with the guanosine deletions replicated slower, while the phenotype with the guanosine additions replicated faster. The goal is to have the healthy mitochondrial DNA replicate faster than any contaminating diseased mitochondrial DNA.

Dr. Mitalipov and colleagues concluded that recipient eggs should always be checked to avoid the guanosine deletions; otherwise the diseased mtDNA will out replicate the healthy DNA.

“Even though we don't yet have a thorough answer for every type of combination, the beginning is there,” Dr. Mitalipov explained. “We can say that there is a phenomenon of reversal, and it looks like to avoid it you would have to somehow match not [necessarily] the entire mtDNA molecule. [We have learned that the] regulatory region is more important than anything else.”

While Dr. Mitalipov acknowledged that more work remains to be done, he believes that haplotype matching is an important next step in bolstering MRT efficacy.

“This happens in vitro, in an artificial system — it could be a plain in vitro effect, and may never happen to children, but we want to be careful and so decided we have to take this into account,” he said.

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Experts in mitochondrial diseases in children said the study provides a major step forward in advancing mitochondrial replacement therapies, but they noted certain limitations and challenges ahead. “Mitochondrial diseases are devastating, and currently there are no US Food and Drug Administration-approved treatments to improve mitochondrial function,” said Bruce H. Cohen, MD, FAAN, professor of pediatrics at Northeast Ohio Medical University and director of the NeuroDevelopmental Science Center at Akron Children's Hospital.

“Treatments are limited to symptomatic therapy for the epilepsies, and for the diabetes and the heart disease,” he continued. “But there's no treatment for the blindness, for the dementia, among other disorders.” He said that while MRT is not a treatment for mitochondrial disease, it is an unprecedented approach for families carrying pathogenic mitochondrial mutations to have healthy children.

Dr. Cohen noted that only one out of seven instances of mitochondrial disease is maternally inherited. For those who carry mutations in Mendelian fashion, MRT would not be applicable.

That said, Dr. Cohen pointed to the study's proposal of a donor-mother compatibility paradigm as one of its most significant advances.

“This study is a great accomplishment because the critics of this technology have said ‘You haven't figured out a way to ensure that the mutated mitochondrial DNA doesn't re-replicate,’” he explained. “This takes us one step closer to understanding what the actual issue may be, and the preventative steps that would need to take place.”



“The paper supports strongly a previous study [published in Nature in 2013] by Dr. Michio Hirano and Dr. Daniel Pauli here at Columbia, who used the same techniques,” said Salvatore DiMauro, MD, the Lucy G. Moses Professor of Neurology at Columbia University Medical Center. “While the previous paper worked with healthy maternal mtDNA, this study's use of mutant mitochondrial DNA made it novel.”

“This is a tremendous precaution for protecting this technique,” Dr. DiMauro said. “And I think it is going to start to be used with approval by the government in some countries.”

Eric A. Shoubridge, PhD, chair of McGill University's department of human genetics and author of the editorial accompanying the Nature study, said MRT, which is only legal in the United Kingdom, faces other difficulties.

“It's technically demanding — more demanding than other assisted reproductive technologies,” he said. “So there probably aren't going to be that many centers that will be licensed to carry it out.”

“The only worry is if there's an essentially selfish genome, which can take over because it outcompetes the rest,” Dr. Shoubridge said. “Replicative advantage in some mitochondrial DNA is something that has to be taken into account when considering a donor.”

Deepening our understanding of these potentially “selfish” genomes would be an important next step, Dr. Shoubridge said, adding that animal models would probably be more useful than human embryonic stem cell lines for testing haplotype combinations.

“The problem with working with human embryos is, of course, you can only take them to the blastocyst stage [legally], so you can't study what happens afterwards,” he said. “That's why I suggest it could be done in other vertebrate models, where you would put in other mitochondrial DNA haplotypes and find out whether the replication differences they found in human mitochondrial DNA haplotypes allowed one type to take over in a post-implantation embryo.”

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