Exchanging Cellular Powerhouses

Mitochondria are organelles that are often referred to as the powerhouses of our cells. Mitochondria are the sites where ATP molecules are made, and ATP is the energy unit allowing many processes in the cells to take place- replication, signalling, cell death and many other pathways require ATP. Mitochondria are also the only organelles in our cells which have their own genome, therefore, all our cells actually have two genomes- the nuclear genome (which is what people usually refer to when they talk about cellular genome) and the mitochondrial genome. The mitochondrial genome is much smaller that the nuclear one. The human nuclear genome is has around 3 billion base pairs, which are all contained in 23 chromosome, whereas the mitochondrial genome has only 16 569 base pairs and is maintained as a circular chromosome of a double-stranded DNA.

Mitochondrion
Mitochondrion

Unlike the nuclear genome, half of which comes from the father and half of which comes from the mother, the mitochondrial genome is inherited uniquely from the mother. When a sperm fertilises an egg, any sperm-derived mitochondria are quickly destroyed and only the sperm’s nucleus survives. Because the mitochondrial genome has just a single inheritance line, it has also been used to date back the earliest genetic female ancestor, so called mitochondrial Eve. The mitochondrial Eve has been estimated to have lived around 200,000 years ago in Africa, which is also considered to be one of the proofs for the out of Africa migration of the earliest human species.

When the mitochondria divide inside the cells their genomes need to be replicated as well. However, like any replication process, mitochondrial genome replication is not a foolproof process. Genetic mutations can occur inside mitochondrial genome, which, just like the mutations in the nuclear genes, may have detrimental effects on cellular processes. The first mitochondria-linked disease has been described in 1988 and it is possible that as many as 1 in 6000 people are at risk of developing some form of mitochondria-linked disease. Some of the mitochondrial diseases have been well characterised and can be traced using genetics. It is often the case that the same person will have a mixture of mitochondrial genomes that some of which contain and some of which lack the mutations conferring a disease and the severity of the disease is determined by the ratio of the two types. The fact that one cell can have several mitochondrial genomes means that lethal or deleterious mutations in them can persists at low frequencies without being eliminated from the population (somewhat like the recessive alleles). If, during fertilisation, the egg that is being fertilised contains many of these defective genomes it means that the newborn will also most likely inherit the associated genetic disease.

Over the past several decades methods have been developed that allow to potentially avoid the inheritance of defective mitochondrial genomes. Referred to as Mitochondrial Replacement Therapies (MRTs), these methods in a way exploit the fact that all mitochondria are maternally inherited. Therefore, as long as that at the point of fertilisation the mitochondria in the egg have ‘healthy’ genomes the mutations will not be passed on. So, two options are possible: infusion of eggs with mitochondria that contain healthy genomes in a hope of outcompeting the defective ones contained in the egg, or transferring the chromosomes (i.e. the egg’s nuclear genetic material) to a donor egg, which lacks its own nucleus but has healthy mitochondrial genes. The latter method has proven to be more effective and it is called meiotic-spindle transfer (MST). The video below shows how MST works.

Meiotic-spindle transfer
Meiotic-spindle transfer

Firstly, the spindle-chromosomal complex (basically a nucleus, which lacks the nuclear membrane) is isolated from a donor egg (e.g. a mother that carries mitochondrial with a disease-associated mutations). This is done using a tiny pipette, which is inserted into an egg with a laser assisted drilling. The pipette captures the spindle-chromosomal complex as well as a tiny bit of the egg’s cytoplasm and membrane. The spindle, cytoplasm and the membrane form tiny lipid droplet-like structure called karyoplast. The karyoplast is then mixed with an extract from a Sendai virus preparation. The trick here is that the Sendai virus extract contains special viral proteins that induce membrane fusion. The virus uses these proteins to enter cells by inducing the fusion between cellular membrane and viral envelope. However, in MST procedure the extract lacks any “live” virus or even its genome, it only contains the viral proteins. Consequently, incubating a karyoplast with Sendai virus extract for a few minutes causes the viral proteins to bind to the karyoplast. This karyoplast is then placed next to the recipient egg (containing no nucleus of its own but with healthy mitochondrial genomes) and the viral proteins mediate membrane fusion, which allows the nucleus of the donor cell to enter the recipient egg. And voilà, you have an egg with mothers nuclear genome, no deleterious mutations in the mitochondria and it can now be in vitro fertilised with the father’s sperm.

The first experiments describing MST used Rhesus macaque eggs and resulted in the first animals known to be produced by the spindle–chromosomal complex transfer procedure. No obvious developmental or physical abnormalities were observed in these macaques. MST is, however, still not widely used clinics for several reasons. Firstly, the technique is quite complicated and available only in limited number of places. Secondly, there are still concerns about a potential carryover of donor mitochondria to the recipient egg. When the karyoplast is formed it contains a small volume of donor cytoplasm and that cytoplasm can potentially contain mitochondria. Indeed, reports have shown that typically less than 2% of mitochondria in a recipient egg will end up being of donor egg origin. While this is a small number, it has recently been shown that these defective donor mitochondria may have some selective advantages allowing them to replicate faster than the good genomes-containing recipient mitochondria. Consequently, during the development the embryonic stem cells that originate from the fertilised egg may end up having a large proportion of defective mitochondrial genomes. With all of these concerns the specific MRT procedures are currently only licenced in UK, even though it is possible to get the procedure in countries where certain exceptions can be made (see a recent headline here).

The first animals born using meiotic-spindle transfer procedure
The first animals born using meiotic-spindle transfer procedure

Tachibana, Masahito, et al. “Mitochondrial gene replacement in primate offspring and embryonic stem cells.” Nature 461.7262 (2009): 367-372.

Kang E, Wu J, et al. Mitochondrial replacement in human oocytes carrying pathogenic mitochondrial DNA mutations. Nature. Macmillan Publishers Limited, part of Springer Nature. All rights reserved; advance online publication SP – EP .

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