Patricia F. Fitzpatrick Dimond Ph.D. Technical Editor of Clinical OMICs President of BioInsight Communications

Focusing on Muscles, Nerves, and the Brain

Heritable mitochondrial diseases vary in symptoms and severity, most commonly including developmental delays, seizures, fatigue, muscle weakness, vision loss, and heart problems, and in some cases premature death.

Most mitochondrial disorders known to date are inherited in either an autosomal recessive manner or via maternal germline transmission. Therapeutic options for most patients with mitochondrial DNA (mtDNA) disease remain limited to management of treatable symptoms, such as in epilepsy, cardiac disease, and diabetes. For maternally inherited disease, researchers have focused on preventing transmission through technically challenging mitochondrial replacement (MRT) techniques that have provoked considerable regulatory discussion.

Mitochondria carry their own multicopy genome. Human mtDNA is a circular, double-stranded, supercoiled molecule present in hundreds to several thousands of copies per cell. As the sole constitutive extrachromosomal DNA in human cells, mtDNA comprises a small genome (16.6 kb) with only 37 genes, 13 of which encode proteins and 24 encode transfer and ribosomal RNA. All are involved with the synthesis of proteins that form key subunits of the mitochondrial respiratory chain.

The mitochondrial genome occurs in multiple copies in all cells. Mitochondrial genomic changes occur as point mutations and deletions, and many hundreds of different mutations associated with disease have been described.

Mitochondria and their DNA are inherited solely from the mother, because the paternal mtDNA present in the sperm is destroyed following fertilization of the egg. In almost all patients affected by mitochondrial disease, mutated mtDNA coexists with wild-type mtDNA, a condition referred to as mtDNA heteroplasmy.

Heteroplasmy plays a key role in determining the clinical severity of mitochondrial diseases because mitochondrial function is affected when a relatively high number—usually >70–80%—of mutated mtDNA compared to wild type (wt) exists. Heteroplasmy can be dynamic, changing during the lifetime in both mitotic and postmitotic tissues, due to the cell cycle–independent mtDNA replication. Defective mitochondria cause most damage in the body tissues and organs that consume the most energy—muscles, nerves, and the brain.

Studies in mice developed to harbor mitochondrial gene mutations have demonstrated the direct cause-and-effect relationship between mitochondrial dysfunction and disease. Mutations in nuclear DNA (nDNA)-encoded mitochondrial genes involved in energy metabolism, antioxidant defenses, apoptosis via the mitochondrial permeability transition pore (mtPTP), mitochondrial fusion, and mtDNA biogenesis have already demonstrated the phenotypic importance of mitochondrial defects.

Modeling of mtDNA changes has been approached by either engineering nuclear genes or by direct manipulation of mtDNA. Mouse models with mtDNA mutations have been created by genetic modification of nDNA-encoded genes involved in mtDNA maintenance, like POLG, Tfam, and Twinkle. Specific deletions have been created in mtDNA by the introduction of defective mitochondria into mouse zygotes or by the expression of mitochondria-targeted restriction endonucleases.

Overcoming Technical Issues

Despite technical barriers and ethical considerations, investigators are attempting techniques to either remove mutated mtDNA by specifically targeted restriction endonucleases or by inhibiting replication of mutated mtDNA, with the goal of reducing heteroplasmy to favor functional mtDNA. Approaches include pronuclear transfer using a mitochondrial donor zygote and a metaphase II (MII) spindle transfer technique using a mitochondrial donor oocyte, followed by intracytoplasmic sperm injection fertilization. Both techniques involve monitoring embryo development following transfer. Researchers don’t expect that these MRTs will prove to be 100% effective at depleting the abnormal mtDNA. They anticipate only up to 1–2% of the original amount would remain, a level below that which is likely to cause disease.

Pradeep Reddy, Ph.D., and colleagues at the Gene Expression Laboratory, Salk Institute for Biological Studies in La Jolla, reported in Cell (“Selective Elimination of Mitochondrial Mutations in the Germline by Genome Editing,” Cell 161:459–469, that they had developed a strategy for preventing germline transmission of mitochondrial diseases by inducing mtDNA heteroplasmy shift through the selective elimination of mutated mtDNA.

As a proof of concept, the investigators used NZB/BALB heteroplasmic mice that contain two mtDNA haplotypes, BALB and NZB, and selectively prevented their germline transmission using either mitochondria-targeted restriction endonucleases or transcription activator-like effector nucleases (TALENs).

To test the potential use of these customizable mito-TALENs in humans, the group generated artificial mammalian oocytes through fusing mouse oocytes with the cells of patients with two different mitochondrial diseases—either Leber’s hereditary optic neuropathy (LHOND) or neurogenic muscle weakness with ataxia and retinitis pigmentosa (NARP). Using specifically designed mito-TALENs, the group effectively reduced both LHOND- and NARP-specific mtDNA in separate experiments, suggesting that this technology might prevent germline transmission of mitochondrial disease.

Writing in Nature (“Mitochondrial Gene Replacement in Primate Offspring and Embryonic Stem Cells,” Nature 461, 367–372, doi:10.1038/nature08368), Masahito Tachibana, Ph.D.,  and colleagues at Oregon National Primate Research Center, Oregon Stem Cell Center and the Departments of Obstetrics and Gynecology and Molecular and Medical Genetics, Oregon Health and Science University, said they had demonstrated that the mitochondrial genome can be replaced in mature non-human primate (Macaca mulatta) oocytes by spindle–chromosomal complex transfer from one egg to an enucleated, mitochondrial-replete egg.

The reconstructed oocytes with the mitochondrial replacement were capable of supporting normal fertilization and embryo development and produced healthy offspring. Genetic analysis confirmed that nDNA in the three offspring born so far originated from the spindle donors, whereas mtDNA came from the cytoplast donors. No contribution of spindle donor mtDNA was detected in offspring.

The authors said their studies show that spindle replacement may provide “an efficient protocol” for replacing the full complement of mitochondria in newly generated embryonic stem cell lines. This approach may offer a reproductive option to prevent mtDNA disease transmission in affected families.

Preventing Disease Transmission

The theoretical goal of these techniques is to prevent disease transmission by creating an embryo with nDNA from the intended mother and mtDNA from a woman with nonpathogenic mtDNA through modification of either an oocyte (egg) or zygote (fertilized egg). Although MRT could provide a previously unavailable reproductive option, the National Academies of Science committee that authored a 2016 report “Mitochondrial Replacement Techniques: Ethical, Social, and Policy Considerations”  ( considering both techniques and ethical aspects of concluded that “MRTs would have no health benefits for people who already have mtDNA diseases, nor would they prevent the occurrence of newly arising mtDNA mutations.”

In its deliberations, the committee considered both maternal spindle transfer (MST) involving manipulation of oocytes and pronuclear transfer (PNT) involving manipulation of zygotes. If effective, the committee said MRT “could satisfy the desire of women seeking to have a genetically related child without the risk of passing on mtDNA disease, but the techniques raised ethical, social, and policy issues.”

Researchers working at the Wellcome Trust Centre for Mitochondrial Research at Newcastle University said in the New England Journal of Medicine (“Mitochondrial Donation—How Many Women Could Benefit?” N Engl J Med 372:885–887, that because inherited mutations in mtDNA cause genetic diseases for which there is no effective treatment, new techniques based on in vitro fertilization (IVF), including pronuclear and MII spindle transfer, have the potential to prevent the transmission of serious mtDNA diseases.

Wellcome Trust researchers also noted that they will be the first to offer mitochondrial donation if Parliament agrees to new regulations regarding enforcement of the Human Fertilization and Embryology Act (1990). In February 2015, U.K. lawmakers approved the use of mitochondrial replacement to prevent the transmission of mitochondrial diseases.

This technology depends on a form of IVF involving the transfer of the nuclear genome from one patient’s embryo into donor embryos with healthy mitochondria. The approval made the U.K. the first country that explicitly allows the procedure, which would combine DNA from two biological parents and an egg donor.

The technique will be allowed under “fairly tight regulation.” Researchers planning on offering the service to couples still must apply for and receive a license from the country’s Human Fertilization and Embryology Authority. In essence, the law allows women to have genetically related children who don’t carry mitochondrial mutations. But the technique remains controversial because it modifies the DNA of an embryo in a way that could be passed on to future offspring and generations.

The Wellcome group cited the medical need for innovative procedures to prevent mitochondrial disease, noting that 2473 women in the U.K. and 12,423 women in the U.S., aged between 15 and 44 years, are at risk of passing on potentially lethal mitochondrial DNA disease to their children, amounting to about 152 births per year in the U.K. and 778 births per year in the U.S.

The group concluded, however, that to ensure that clinical investigations of MRT were performed ethically, certain conditions and principles would need to govern the conduct of clinical investigations and potential future implementation of MRT. 

In the U.S., no federal law bans human embryo research, but funding restrictions exist. Federal funding under the Dickey–Wicker amendment prohibits the creation of human embryos for research purposes or research in which a human embryo is harmed or destroyed. As might be expected, reproductive scientists say that funding and regulatory barriers pose the risk that the U.S. will lag behind other countries such as the U.K. in this important area of research and novel therapeutics.


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