Mitochondria are fascinating. These tiny organelles that reside within almost all of the cells in our bodies (mature red blood cells being an exception) generate the supply of a molecule called adenosine triphosphate (ATP) which is the principle source of energy that cells, and ultimately ourselves, need to survive. They also have an intriguing evolutionary history, being descended from bacteria that over one and a half billion years ago formed a symbiotic relationship with primitive eukaryotic cells that are the ancestors of today’s plants and animals. A legacy of this ancestry is that animal mitochondria contain a tiny genome that encodes 37 genes that are crucial to the mitochondria’s function, and separate from the main genome which is found in the nucleus of the cell and contains just over 20,000 coding genes. Unlike nuclear genes, half of which are inherited from our mother and half from our father, mitochondrial genes are almost always inherited from the mother only, which means that if a mother has a mitochondrial genome mutation it will always be passed on to her children. However, since a human egg cell contains many mitochondria, and only some of them may be defective, there is usually a threshold level of defective mitochondria which must be reached before the defects cause disease in children, and the severity of disease can be very variable. Nevertheless inherited mitochondrial disorders affect as many as 4,000 children born in the USA every year, and for almost all of them treatment options are limited.
One way in which the transmission of mitochondrial diseases can be prevented is by screening embryos during IVF, and earlier this year we reported on how a team at the Oregon National Primate Research Centre (ONPRC) led by Dr. Shoukhrat Mitalipov discovered through studies performed on Rhesus macaques how to improve the efficiency of this screening. However in cases where screening does not identify eggs that are free from mitochondrial genetic defects other ways of preventing transmission of the disorders are being examined, and one of these is the possibility of replacing the damaged mitochondria with healthy mitochondria from a donor.
Yesterday in a publication in Nature (1), Dr. Mitalipov’s team at ONPRC announced another major advance made possible through research on Rhesus monkeys, the first demonstration that it is possible to replace the faulty mitochondria of a human egg cell before fertilization and create healthy looking human embryos, from which embryonic stem cells could be derived that were identical to controls created through normal IVF.
Briefly, the procedure involved the removal of the nuclear genetic material from the egg of a patient whose mitochondrial DNA contains mutations, and its transplantation into an egg containing normal mitochondrial DNA from which the nuclear genetic material has been removed. More detailed descriptions and discussion of the process used in this therapy and the team’s results can be found on the Oregon Health and Science University website, reports on the BBC and LA Times, and in Nature News, it’s clearly been a study that has caught the imagination of a lot of people! It’s worth noting that a child born after fertilization with the partner’s sperm would be free of risk from maternal mtDNA mutations as well as being the biological child of the patients, since the mitochondrial genome accounts for only 37 of over 20,000 coding genes in the body it is inaccurate to refer to these as 3 parent embryos.
The news reports make it very clear that research on monkeys was crucial to this advance, indeed the potential of the technique used – which they term spindle–chromosomal complex transfer – was first demonstrated when they were able to produce 3 healthy monkey infants in 2009 (2). They started by examining the distribution of Rhesus monkey mitochondria during the process of meiosis – the type of cell division through which gametes (sperm and egg cells) are produced – using confocal laser scanning microscopy, and observed that at a particular late stage in the process termed the metaphase II stage the mitochondria were distributed relatively uniformly throughout the cytoplasm, except immediately around the chromosomes and the spindle apparatus (a protein structure segregates chromosomes between daughter cells during cell division), which were devoid of mitochondria. This suggested that it might be possible to isolate the spindle–chromosomal complex at this stage and transfer it to an egg cell from which the egg had been removed without transferring any mitochondria at the same time.
A significant challenge was how to avoid damaging the spindle-nuclear complex during this operation, but the team had recently developed new techniques to transfer the nuclei of adult skin cells from monkeys into egg cells and successfully derive embryonic stem cells from the resulting clones. By modifying these techniques they were able to reconstructed eggs that were capable of being fertilized normally, undergoing embryo development and producing healthy offspring. Genetic analysis confirmed that nuclear DNA in the three infant macaques originated from the spindle donors whereas mitochondrial DNA came from the cytoplast donors. This set the stage for the work announced yesterday.
In addition to reporting the production of human embryos through spindle–chromosomal complex transfer (ST) this week’s Nature paper (1) also reported the outcome of follow-up examination from birth to 3 years of four monkeys born through ST – the 3 reported in 2009 and one born subsequent to that publication – and found that they were developing normally and were in good health.
Because egg cells only remain viable for a short period of time after they are harvested from a donor, it is considered crucial that ST can be performed successfully using frozen egg cells for this technique to be clinically viable, so the team also examined if it was possible to do this using thawed Rhesus macaque cells. They were successful; the experiment resulted in the birth of a healthy monkey. More surprisingly they also found to their surprise that while the spindle–chromosomal complex could withstand prior cryopreservation the technique failed when the egg into which the spindle-chromosal complex is transferred had been frozen – indicating that most of the damage to the frozen egg is to its cytoplasm, rather than to the nucleus as had previously been thought, a discovery that may have wider implications for the future improvement of human egg cryopreservation and IVF techniques.
Impressive as this study is it is by no means the end of the road, this technique needs further refinement and optimization before anyone should attempt to use it in the clinic, but it does provide both scientists and ethicists with very valuable information. This is particularly true in the UK, where the Human Fertilisation and Embryology Authority is reviewing this technique and another that is being developed by Professor Mary Herbert at the University of Newcastle. Speaking to the BBC yesterday, Peter Braude, Professor of Obstetrics and Gynaecology at King’s College London, said:
It is exactly the sort of science that the HFEA expert committee recommended needed doing, and demonstrates further the feasibility of this technique.”
We at Speaking of Research congratulate Dr. Mitalipov and his team at ONPRC on their groundbreaking work.
1) Masahito Tachibana, Paula Amato, Michelle Sparman, JoyWoodward, Dario Melguizo Sanchis, Hong Ma, Nuria Marti Gutierrez, Rebecca Tippner-Hedges, Eunju Kang, Hyo-Sang Lee, Cathy Ramsey, Keith Masterson, David Battaglia, David Lee, Diana Wu, Jeffrey Jensen, Phillip Patton, Sumita Gokhale, Richard Stouffer& Shoukhrat Mitalipov “Towards germline gene therapy of inherited mitochondrial diseases” Nature Published online 24 Oct 2012, doi:10.1038/nature11647
2) Masahito Tachibana, Michelle Sparman, Hathaitip Sritanaudomchai, Hong Ma, Lisa Clepper, Joy Woodward, Ying Li, Cathy Ramsey, Olena Kolotushkina & Shoukhrat Mitalipov “Mitochondrial gene replacement in primate offspring and embryonic stem cells” Nature 461, 367-372 (2009) doi:10.1038/nature08368