Mitochondrial Replacement May Require Matching, Like Transplants

For mitochondrial replacement techniques to prevent transmission of disease, the genetic backgrounds of mitochondrial (mt) DNA between mother and donor may need to match, according to results of a study published today in Nature.

Shoukhrat Mitalipov, PhD, Director of the OHSU Center for Embryonic Cell and Gene Therapy at Oregon Health & Science University and colleagues used the spindle nuclear transfer approach on eggs from four women who had children with Leigh syndrome and one woman who had three children with MELAS. This strategy takes the mother's egg nucleus at the stage of division (meiosis) when it is clinging to the spindle fibers (which separate chromosomes) and places it into an egg cell from a donor that has had its nucleus removed. The resulting cell has the mother’s nuclear DNA but mostly the donor’s normal mtDNA.

Add sperm and what results is a fertilized ovum with healthy mtDNA. And in the experiments that’s what happened, with up to 99% of the mtDNA being from the donor – at first. But further experiments showed that if the donor mtDNA is missing a single base, and the maternal mtDNA isn’t, the donor DNA can’t replicate efficiently. Over several division cycles, this incompatibility results in maternal DNA – bearing the mutation meant to be weeded out – instead taking over. (The one-base deletion is in the D-loop region of the mtDNA, in a section called the CSBII, for conserved sequence box II. It does not encode protein, but controls DNA replication rate.)
The researchers demonstrated this unexpected takeover, which they term an “mtDNA reversion,” in embryonic stem (ES) cells that they cultured from day-5 blastocysts derived from the fertilized ova. (This stage of prenatal development looks like a hollow ball with a smear of cells on the interior face; the smear develops into the embryo, the surrounding cells into the extra-embryonic membranes.) They also showed that the reversion persists when the ES cells divide to give rise to specialized daughter cells (heart muscle cells, neural progenitor cells, and teratomas).

The concern is the possibility of reversion if mitochondrial transfer is used to conceive offspring free of the mitochondrial disease that the mother carries. The reversal wasn’t seen in experiments using rhesus monkeys or mice.

Co-author Paula Amato, MD, described the findings in a news conference. “These eggs underwent comparable fertilization rates to IVF controls. The process was highly efficient, with less than 1% carryover of mutant mitochondrial DNA. However, some of the ES cells did revert back to mutant mtDNA, so we looked at potential mechanisms. We’ve had long- standing concerns about possible mitochondrial/nuclear DNA mismatch from animal models.”

Thinking ahead, Dr. Amato added, “we propose a matching paradigm to increase safety of the technology when we move to clinical trials. It would be analogous to HLA matching used in organ transplants, but wouldn’t necessarily want identical HLA types but haplotypes (linked genetic markers) that have a replicative advantage”.

Dr. Mitalipov added that they must determine whether the reversion arises in the unnatural culture environment of ES cells but not necessarily in the body. They must also distinguish between an incompatibility between the maternal and donor D-loop variant, or just the presence of a particular D-loop gene variant in causing the reversion. “It may be an independent mechanism to how some mtDNA overcompetes and the cells grow faster and take over embryonic development.” He calls for additional studies “so we can completely avoid this phenomenon.

An accompanying News and Views by Eric A. Shoubridge, from the Department of Human Genetics, at the Montreal Neurological Institute of McGill University, summarizes the challenges of developing mitochondrial replacement technology: It’s technically difficult, costly, and is only approved in the UK.

The new findings introduce yet a new wrinkle: the potential to reversion to the mutation.

It might make more economic sense to focus on developing treatments for people who have mitochondrial disease, or to support research to develop technology to select embryos that have not reverted – unfortunately reversion isn’t apparent until after the blastocyst stage.

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