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Thoughts & Opinion
N. Gemmell and J. N. Wolff
Commentary
Mitochondrial replacement therapy: Cautiously replace the master manipulator Neil Gemmell1)* and Jonci N. Wolff2) Around 1 in 200 children born each year have mutations in the mitochondrial DNA (mtDNA)—the genome of the cellular powerhouse. In most cases this causes only mild disease, often without any symptoms. However, for about 1 in 6,500 individuals, mitochondrial disease causes serious and often fatal conditions, which include blindness, muscular weakness, and heart failure. Currently available therapies for individuals carrying mitochondrial disease are not curative, but at best supportive in nature. Mitochondrial replacement (MR), via three-way in vitro fertilization (IVF) [1–3], may be a tractable intervention to circumvent what are clearly devastating diseases. The approach differs from traditional IVF in that embryos effectively have three parents who each make a genetic contribution, with the embryo possessing nuclear DNA from the mother and father and mtDNA from a donor female that has healthy mtDNA [1–3]. The key merit of the approach is that it limits the carryover of disease carrying mtDNA, while enabling both the mother and the father to have a genetic connection to their offspring, which would otherwise be lost if standard egg donation was used. These therapies promise a way to reduce the risk of having offspring with potentially devastating mitochondrial disease and affected families eagerly await such treatments. However, MR approaches are technically challenging, have low DOI 10.1002/bies.201500008 1)
2)
Allan Wilson Centre, Department of Anatomy, University of Otago, Dunedin, New Zealand School of Biological Sciences, Monash University, Clayton, Australia
*Corresponding author: Neil Gemmell E-mail:
[email protected]
rates of success [1–3], and are not risk free even if healthy offspring can be produced [4, 5]. Despite growing optimism based on successful mitochondrial replacement experiments [1–3], we remain ignorant of the broad effects that the small mitochondrial genome (mtDNA) might have on phenotype, both directly through its role in cellular energetics and indirectly as a manipulator or moderator of nuclear genetic events [6]. A growing literature shows that mtDNA can have wide effects on phenotypes, and that these effects are particularly prominent in males. Experiments in mice and fruit flies have shown that simply varying the mtDNA, while keeping the nuclear genetics constant, can result in major alterations to longevity, fertility, and behavior [7]. In addition, the long-held view that mtDNA was a fairly minor player on phenotype is currently being drastically overhauled: new work shows that mtDNA haplotype has direct and indirect effects on nuclear gene regulation [6]. Faced with this evidence, it seems prudent to move towards clinical application of mitochondria replacement with caution. One of the key considerations will be to determine whether outcomes from such procedures are affected by the similarity between the donor and recipient mtDNAs. A strong prediction is that as mtDNA donor and recipient mtDNAs diverge, offspring survival and health may be reduced as a consequence of the breakdown in co-evolved mtDNA– nuclear interactions [8]. The literature is already rich with examples of how mitochondrial genome variation maintained within the human population alters cellular physiology and disease susceptibility of carriers depending on the nuclear background alongside which this variation is expressed. Reinforcing these concerns is a recent study
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showing that mismatched mitochondria in nuclear-transfer-derived cells—a technique akin to pronuclear transfer (PNT) suggested for MR—possess alloantigenecity, triggering an immune response in a murine model [5]. Likely, establishing the principle of replacing mutant mtDNA with the most similar non-disease carrying mtDNA is a good one to aspire to, and given advances in sequencing technologies such matching would add a trivial cost to what is a significant manipulation, while likely improving outcomes for both the offspring and their parents. The other issue that requires further thought and experimentation is that a large number of mtDNA disorders appear late in life, and we simply do not yet know what the longer term effects of such replacements might be. While flies, mice, and non-human primates may give us some insights into the utility of such therapies, they are unlikely to help us know what transpires in the later life of humans. One potentially complicating issue here is that mtDNA replacement is not 100%, thus there will be potential for carryover of disease-causing mutant mtDNAs, and these may increase in frequency within an individual over time. Pre-clinical trials applying MR therapies to mammalian model systems led to embryos containing varying amounts of carryover mtDNAs [1, 2]. Perhaps the most comprehensive estimate comes from a recent comparative study applying PNT, spindle-chromosome transfer (ST), and polar body transfer (PBT1, PBT2) in parallel to a mouse model [2]. Probing for heteroplasmy (the presence of more than one mtDNA type in a cell) in F1 infants (tail tips), this study found an average carryover of over 5% for ST individuals, over 20% for PNT individuals, and 0 (i.e. below detection limit of 1%) to 2% for
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Commentary
N. Gemmell and J. N. Wolff
PBT1 and PBT2 individuals, respectively [2]. Importantly, the authors extended their experiments to examine tissues high in energy demand (brain, heart, lung, liver, kidney) across F1 infants, and further examined F2 infants (toe clips). These additional experiments showed that segregation of disease mtDNA during early stages of embryogenesis led to varying degrees of heteroplasmy across examined tissues for ST (0–6.88%), PNT (5.55– 39.8%), and PBT2 (0–3.62%), whereas heteroplasmy levels for PBT1 remained below the detection limit of 1% for all individuals and organs tested. Moreover, experiments on F2 infants showed that mutant mtDNA had also entered the F1 germ line, and led to the intergenerational maintenance—or even increase—of heteroplasmy levels for PBT2, ST, and PNT. Even though no heteroplasmy was detected within PBT1 individuals, its presence below 1% could not be excluded. While in some cases, contributions of mutant mtDNA in examined tissues may lie well below thresholds above which mitochondrial disease is typically expressed, more research is needed to understand better the quantitative genetics of mtDNA replication and segregation within cellular subpopulations, tissues, and organs over an individual’s lifetime. What work there is suggests that mtDNA frequencies can alter significantly between tissue types [2], and we may find that the disease-causing form may possess some replicative or even selective advantage, leading it to increase in frequency over time even after successful replacement interventions. Among such affected tissues, the primordial germ cells that give rise to the oocyte in female offspring are a particular concern. Although subject to ongoing debate and research efforts, the general view is that mtDNA numbers are profoundly reduced in these cells, going through a bottleneck of around 100 and perhaps as few as 35 molecules in human [9]. This bottleneck is predominantly a stochastic or random process, although purifying selection, a process
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Thoughts & Opinion
by which deleterious mutations are removed during germ line development, may act as safeguard against the inheritance of mutant alleles. This safeguard however seems inefficient, as mitochondrial diseases are often traceable over multiple generations. If inherited, the stochasticity of the mtDNA replication process may therefore result in the mutant mtDNA arising at high frequency in the germ cell precursors, in turn leading to frequent and drastic intergenerational heteroplasmy shifts, such as those recently reported in a cohort of 39 mother–child pairs [9]. Thus, it is not impossible to imagine that despite therapeutic treatments in early life, daughter offspring may end up being potential carriers of these devastating diseases, just as their (main) biological mothers were before them. In addition, mitochondrial replacement almost universally creates a situation of heteroplasmy, which itself may lead to complications with potential costs to offspring fitness and health [10]. Indeed, concerns about heteroplasmy, led to the US Food and Drug Administration to ban an earlier form of mitochondrial replacement treatment, ooplasmic transfer (OT). In OT, the cytoplasm of an oocyte carrying good mtDNA is injected into the oocyte of an individual carrying mtDNA with disease causing mutations; a process that may lead to substantial carryover of the dysfunctional mtDNA. While heteroplasmy rates for three-way IVF therapies are much lower than those observed in ooplasmic transfer, the risks associated with heteroplasmy, the effects of which remain predominantly unknown, persist [10]. This additional risk factor may require particular attention when it comes to the age of mitochondrial donors, because the number of heteroplasmies observed in offspring is expected to be positively associated with maternal age at the point of fertilization [9]. While there is definite promise in mitochondrial replacement therapy, the suggestion made in the popular
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press that this process ‘‘is just like changing the battery of a laptop or camera’’ fails to capture the complexity of the problem. A better analogy might be to consider this akin to replacing one of the CPUs in your laptop or camera: mitochondria certainly have functions well beyond any battery of which we are aware, and a precautionary principle should be applied in their replacement. The authors have declared no conflicts of interest.
References 1. Craven L, Tuppen HA, Greggains GD, Harbottle SJ, et al. 2010. Pronuclear transfer in human embryos to prevent transmission of mitochondrial DNA disease. Nature 465: 82–5. 2. Wang T, Sha H, Ji D, Zhang HL, et al. 2014. Polar body genome transfer for preventing the transmission of inherited mitochondrial diseases. Cell 157: 1591–604. 3. Tachibana M, Amato P, Sparman M, Woodward J, et al. 2013. Towards germline gene therapy of inherited mitochondrial diseases. Nature 493: 627–31. 4. Reinhardt K, Dowling DK, Morrow EH. 2013. Mitochondrial replacement, evolution, and the clinic. Science 341: 1345–6. 5. Deuse T, Wang D, Stubbendorff M, Itagaki R, et al. 2015. SCNT-derived ESCs with mismatched mitochondria trigger an immune response in allogeneic hosts. Cell Stem Cell 16: 33–8. 6. Horan MP, Gemmell NJ, Wolff JN. 2013. From evolutionary bystander to master manipulator: the emerging roles for the mitochondrial genome as a modulator of nuclear gene expression. Eur J Hum Genet 21: 1335–7. 7. Wolff JN, Gemmell NJ. 2013. Mitochondria, maternal inheritance, and asymmetric fitness: why males die younger. BioEssays 35: 93–9. 8. Wolff JN, Ladoukakis ED, Enriquez JA, Dowling DK. 2014. Mitonuclear interactions: evolutionary consequences over multiple biological scales. Philos Trans R Soc Lond B Biol Sci 369: 20130443. 9. Rebolledo-Jaramillo B, Su MS-W, Stoler N, McElhoe JA, et al. 2014. Maternal age effect and severe germ-line bottleneck in the inheritance of human mitochondrial DNA. Proc Natl Acad Sci USA 111: 15474–9. 10. Sharpley MS, Marciniak C, Eckel-Mahan K, McManus M, et al. 2012. Heteroplasmy of mouse mtDNA is genetically unstable and results in altered behavior and cognition. Cell 151: 333–43.
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