Bridging the gap between in vitro and in vivo models: a way forward to clinical translation of mitochondrial transplantation in acute disease states.

Mitochondrial transplantation and transfer are being explored as therapeutic options in acute and chronic diseases to restore cellular function in injured tissues. To limit potential immune responses and rejection of donor mitochondria, current clinical applications have focused on delivery of autologous mitochondria. We recently convened a Mitochondrial Transplant Convergent Working Group (CWG), to explore three key issues that limit clinical translation: (1) storage of mitochondria, (2) biomaterials to enhance mitochondrial uptake, and (3) dynamic models to mimic the complex recipient tissue environment. In this review, we present a summary of CWG conclusions related to these three issues and provide an overview of pre-clinical studies aimed at building a more robust toolkit for translational trials.

Mitochondrial transplantation: A promising therapy for mitochondrial disorders.

As a vital energy source for cellular metabolism and tissue survival, the mitochondrion can undergo morphological or positional change and even shuttle between cells in response to various stimuli and energy demands. Multiple human diseases are originated from mitochondrial dysfunction, but the curative succusses by traditional treatments are limited. Mitochondrial transplantation therapy (MTT) is an innovative therapeutic approach that is to deliver the healthy mitochondria either derived from normal cells or reassembled through synthetic biology into the cells and tissues suffering from mitochondrial damages and finally replace their defective mitochondria and restore their function. MTT has already been under investigation in clinical trials for cardiac ischemia-reperfusion injury and given an encouraging performance in animal models of numerous fatal critical diseases including central nervous system disorders, cardiovascular diseases, inflammatory conditions, cancer, renal injury, and pulmonary damage. This review article summarizes the mechanisms and strategies of mitochondrial transfer and the MTT application for types of mitochondrial diseases, and discusses the potential challenge in MTT clinical application, aiming to exhibit the good therapeutic prospects of MTTs in clinics.

Reduction of mtDNA heteroplasmy in mitochondrial replacement therapy by inducing forced mitophagy.

Mitochondrial replacement therapy (MRT) has been used to prevent maternal transmission of disease-causing mutations in mitochondrial DNA (mtDNA). However, because MRT requires nuclear transfer, it carries the risk of mtDNA carryover and hence of the reversion of mtDNA to pathogenic levels owing to selective replication and genetic drift. Here we show in HeLa cells, mouse embryos and human embryos that mtDNA heteroplasmy can be reduced by pre-labelling the mitochondrial outer membrane of a donor zygote via microinjection with an mRNA coding for a transmembrane peptide fused to an autophagy receptor, to induce the degradation of the labelled mitochondria via forced mitophagy. Forced mitophagy reduced mtDNA carryover in newly reconstructed embryos after MRT, and had negligible effects on the growth curve, reproduction, exercise capacity and other behavioural characteristics of the offspring mice. The induction of forced mitophagy to degrade undesired donor mtDNA may increase the clinical feasibility of MRT and could be extended to other nuclear transfer techniques.

Spatiotemporal perturbations of pronuclear breakdown preceding syngamy affect early human embryo development: a retrospective observational study.

PURPOSE: During fertilisation, female and male pronuclei (PNs) migrate to the centre of the ooplasm, juxtapose, and break down synchronously in preparation for the first mitosis. While PN non-juxtaposition and PN breakdown (PNBD) asynchrony are occasionally observed, their developmental implications remain uncertain. This study investigated the possible relationships among the two phenomena, preimplantation development patterns, and live birth rates in single blastocyst transfers. METHODS: A total of 1455 fertilised oocytes cultured in a time-lapse incubator were retrospectively analysed. Fertilised oocytes were divided into four groups according to the presence of PN juxtaposition and breakdown synchrony. The relationships of abnormal PN behaviour with embryo morphokinetics, blastocyst formation, and live birth were evaluated. RESULTS: PN non-juxtaposition and asynchrony were observed in 1.9% and 1.0% of fertilised oocytes, respectively. The blastocyst cryopreservation rates in the synchronous-non-juxtaposed and asynchronous-non-juxtaposed groups were significantly lower than that in the synchronous-juxtaposed group. The rates of clinical pregnancy, ongoing pregnancy, and live birth were comparable among the groups. Non-juxtaposition was significantly associated with increased trichotomous cleavage at the first cytokinesis (P < 0.0001) and an increase in the time interval from PNBD to first cleavage (P < 0.0001). Furthermore, asynchronous PNBD was significantly correlated with increased rapid cleavage at the first cytokinesis (P = 0.0100). CONCLUSION: Non-juxtaposition and asynchronous PNBD is associated with abnormal mitosis at the first cleavage and impaired preimplantation development. However, embryos displaying abnormal PNBD may develop to blastocyst stage and produce live births, suggesting blastocyst transfer as a more appropriate culture strategy.

Mitochondrial Genetic Drift after Nuclear Transfer in Oocytes.

Mitochondria are energy-producing intracellular organelles containing their own genetic material in the form of mitochondrial DNA (mtDNA), which codes for proteins and RNAs essential for mitochondrial function. Some mtDNA mutations can cause mitochondria-related diseases. Mitochondrial diseases are a heterogeneous group of inherited disorders with no cure, in which mutated mtDNA is passed from mothers to offspring via maternal egg cytoplasm. Mitochondrial replacement (MR) is a genome transfer technology in which mtDNA carrying disease-related mutations is replaced by presumably disease-free mtDNA. This therapy aims at preventing the transmission of known disease-causing mitochondria to the next generation. Here, a proof of concept for the specific removal or editing of mtDNA disease-related mutations by genome editing is introduced. Although the amount of mtDNA carryover introduced into human oocytes during nuclear transfer is low, the safety of mtDNA heteroplasmy remains a concern. This is particularly true regarding donor-recipient mtDNA mismatch (mtDNA-mtDNA), mtDNA-nuclear DNA (nDNA) mismatch caused by mixing recipient nDNA with donor mtDNA, and mtDNA replicative segregation. These conditions can lead to mtDNA genetic drift and reversion to the original genotype. In this review, we address the current state of knowledge regarding nuclear transplantation for preventing the inheritance of mitochondrial diseases.

Mitochondrial donation - hope for families with mitochondrial DNA disease.

In 2015, the UK became the first country to approve the use of mitochondrial donation. This novel in vitro fertilisation treatment was developed to prevent transmission of mitochondrial DNA (mtDNA) disease and ultimately give more reproductive choice to women at risk of having severely affected offspring. The policy change was a major advance that surmounted many scientific, legislative and clinical challenges. Further challenges have since been addressed and there is now an NHS clinical service available to families with pathogenic mtDNA mutations that provides reproductive advice and options, and a research study to look at the outcome at 18 months of children born after mitochondrial donation.

Maternal spindle transfer overcomes embryo developmental arrest caused by ooplasmic defects in mice.

The developmental potential of early embryos is mainly dictated by the quality of the oocyte. Here, we explore the utility of the maternal spindle transfer (MST) technique as a reproductive approach to enhance oocyte developmental competence. Our proof-of-concept experiments show that replacement of the entire cytoplasm of oocytes from a sensitive mouse strain overcomes massive embryo developmental arrest characteristic of non-manipulated oocytes. Genetic analysis confirmed minimal carryover of mtDNA following MST. Resulting mice showed low heteroplasmy levels in multiple organs at adult age, normal histology and fertility. Mice were followed for five generations (F5), revealing that heteroplasmy was reduced in F2 mice and was undetectable in the subsequent generations. This pre-clinical model demonstrates the high efficiency and potential of the MST technique, not only to prevent the transmission of mtDNA mutations, but also as a new potential treatment for patients with certain forms of infertility refractory to current clinical strategies.

Infertility is a growing problem that affects millions of people worldwide. Medical procedures known as in vitro fertilization (IVF) help many individuals experiencing infertility to have children. Typically in IVF, a woman's egg cells are collected, fertilized with sperm from a chosen male and grown for a few days in a laboratory, before returning them to the woman's body to continue to develop. However, there are some women whose egg cells cannot develop into a healthy baby after they have been fertilized. Many of these patients use egg cells from donors, instead. This greatly improves the chances of the IVF treatment being successful, but the resultant children are not genetically related to the intended mothers. Previous studies suggested that a cell compartment known as the cytoplasm plays a crucial role in allowing fertilized egg cells to develop normally. A new technique known as maternal spindle transfer, often shortened to MST, makes it possible to replace the entire cytoplasm of a compromised egg cell. This is achieved by transplanting the genetic material of the compromised egg cell into a donor egg cell with healthier cytoplasm that has previously had its own genetic material removed. Using this technique, it is possible to generate human egg cells for IVF that have the genetic material from the intended mother without the defects in the cytoplasm that may be responsible for infertility. However, it is not clear whether this approach would be a safe and effective way to treat infertility in humans. Costa-Borges et al. applied MST to infertile female mice and found that the technique could permanently correct deficiencies in the cytoplasms of poor quality egg cells, allowing the mice to give birth to healthy offspring. Further experiments studied the offspring and their descendants over several generations and found that they also had higher quality egg cells and normal levels of fertility. These findings open up the possibility of developing new treatments for infertility caused by problems with egg cells, so experiments involving human egg cells are now being performed to evaluate the safety and effectiveness of the technique.

Comparative analysis of different nuclear transfer techniques to prevent the transmission of mitochondrial DNA variants.

Prevention of mitochondrial DNA (mtDNA) diseases may currently be possible using germline nuclear transfer (NT). However, scientific evidence to compare efficiency of different NT techniques to overcome mtDNA diseases is lacking. Here, we performed four types of NT, including first or second polar body transfer (PB1/2T), maternal spindle transfer (ST) and pronuclear transfer (PNT), using NZB/OlaHsd and B6D2F1 mouse models. Embryo development was assessed following NT, and mtDNA carry-over levels were measured by next generation sequencing (NGS). Moreover, we explored two novel protocols (PB2T-a and PB2T-b) to optimize PB2T using mouse and human oocytes. Chromosomal profiles of NT-generated blastocysts were evaluated using NGS. In mouse, our findings reveal that only PB2T-b successfully leads to blastocysts. There were comparable blastocyst rates among PB1T, PB2T-b, ST and PNT embryos. Furthermore, PB1T and PB2T-b had lower mtDNA carry-over levels than ST and PNT. After extrapolation of novel PB2T-b to human in vitro matured (IVM) oocytes and in vivo matured oocytes with smooth endoplasmic reticulum aggregate (SERa) oocytes, the reconstituted embryos successfully developed to blastocysts at a comparable rate to ICSI controls. PB2T-b embryos generated from IVM oocytes showed a similar euploidy rate to ICSI controls. Nevertheless, our mouse model with non-mutated mtDNAs is different from a mixture of pathogenic and non-pathogenic mtDNAs in a human scenario. Novel PB2T-b requires further optimization to improve blastocyst rates in human. Although more work is required to elucidate efficiency and safety of NT, our study suggests that PBT may have the potential to prevent mtDNA disease transmission.

Public attitudes towards novel reproductive technologies: a citizens' jury on mitochondrial donation.

STUDY QUESTION: Does an informed group of citizens endorse the clinical use of mitochondrial donation in a country where this is not currently permitted? SUMMARY ANSWER: After hearing balanced expert evidence and having opportunity for deliberation, a majority (11/14) of participants in a citizens' jury believed that children should be able to be born using mitochondrial donation. WHAT IS KNOWN ALREADY: Research suggests that patients, oocyte donors and health professionals support mitochondrial donation to prevent transmission of mitochondrial disease. Less is known about public acceptability of this novel reproductive technology, especially from evidence using deliberative methods. STUDY DESIGN, SIZE, DURATION: This study comprised a citizens' jury, an established method for determining the views of a well-informed group of community members. The jury had 14 participants, and ran over one and a half days in 2017. PARTICIPANTS/MATERIALS, SETTING, METHODS: Jurors were members of the public with no experience of mitochondrial disease. They heard and engaged with relevant evidence and were asked to answer the question: 'Should Australia allow children to be born following mitochondrial donation?' MAIN RESULTS AND THE ROLE OF CHANCE: Eleven jurors decided that Australia should allow children to be born following mitochondrial donation; 7 of whom added conditions such as the need to limit who can access the intervention. Three jurors decided that children should not (or not yet) be born using this intervention. All jurors were particularly interested in the reliability of evidence, licensing/regulatory mechanisms and the rights of children to access information about their oocyte donors. LIMITATIONS, REASONS FOR CAUTION: Jurors' views were well informed and reflected critical deliberation and discussion, but are not intended to be representative of the whole population. WIDER IMPLICATIONS OF THE FINDINGS: When presented with high quality evidence, combined with opportunities to undertake structured deliberation of novel reproductive technologies, members of the public are able to engage in detailed discussions. This is the first study to use an established deliberative method to gauge public views towards mitochondrial donation. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by a University of Sydney Industry and Community Collaboration Seed Award (2017), which was awarded contingent on additional funding from the Mito Foundation. Additional funding was provided by the Mito Foundation. The Foundation was not involved in jury facilitation or deliberation, nor analysis of research data. TRIAL REGISTRATION NUMBER: Not applicable.

Preventing Mitochondrial Diseases: Embryo-Sparing Donor-Independent Options.

Mutant mitochondrial DNA gives rise to a broad range of incurable inborn maladies. Prevention may now be possible by replacing the mutation-carrying mitochondria of zygotes or oocytes at risk with donated unaffected counterparts. However, mitochondrial replacement therapy is being held back by theological, ethical, and safety concerns over the loss of human zygotes and the involvement of a donor. These concerns make it plain that the identification, validation, and regulatory adjudication of novel embryo-sparing donor-independent technologies remains a pressing imperative. This Opinion highlights three emerging embryo-sparing donor-independent options that stand to markedly allay theological, ethical, and safety concerns raised by mitochondrial replacement therapy.

Progress in mitochondrial replacement therapies.

Mitochondrial DNA is maternally inherited, and pathogenic mutations cause a range of life-limiting conditions. Recent studies indicate that transmission of pathogenic mutations may be prevented by reproductive technologies designed to replace the mitochondria in eggs from affected women.

Mitochondrial donation, patient engagement and narratives of hope.

This article develops the sociology of hope and patient engagement by exploring how patients' perceptions and actions are shaped by narratives of hope surrounding the clinical introduction of novel reproductive techniques. In 2015, after extensive public debates, the UK became the first country to legalise a mitochondrial donation technique aimed at preventing the transmission of inherited disorders. The article draws on the accounts of twenty-two women of reproductive age who are at risk of having a child with mitochondrial disease and would be the potential target of the technique. We explore the extent to which our participants engaged with the public debates and how they accounted for their support of mitochondrial donation. We show that while the majority of our participants were in favour of legalisation, they did not necessarily wish to use the technique themselves. We found that hope was multi-faceted, involving hope for self, hope for family and hope for society. We conclude by considering the implications of hope narratives for patients and families and the important but potentially limited role that patients can play as advocates for technology.

Three persons, three genetic contributors, three parents: Mitochondrial donation, genetic parenting and the immutable grammar of the 'three x x'.

In 2015, two novel in vitro fertilisation techniques intended to prevent the inheritance of mitochondrial disease were legalised in the United Kingdom, following an intense period of inquiry including scientific reviews, public consultations, government guidance and debates within the Houses of Parliament. The techniques were controversial because (1) they introduced a third genetic contributor into the reproductive process and (2) they are germline, meaning this genetic change could then be passed down to subsequent generations. Drawing on the social worlds framework with a focus on implicated actors and discursive strategies, this article explores key features of the UK mitochondrial debates as they played out in real time through policy documents and public debate. First, it situates the technology within a repertoire of metaphors, emotional terminology and their politics. It then explores the immutable grammar of 'three x x' that formed a key component of the political debate, by focusing on how institutional reviews discursively negotiated uncertainty around genetic parentage and how beneficiaries were implicated and rendered distant. Following the 2016 announcement of the first baby born through mitochondrial donation (in Mexico) and several pregnancies (in the Ukraine), we close with a discussion about the specific nature of UK regulation within a global economy. Overall, this article contributes to a much needed sociological discussion about mitochondrial donation, emerging reproductive technologies and the cultural significance of genetic material and genetic relatedness.