Term paper: “Mitochondrial replacement

therapy”
 Abstract

Mitochondrial DNA mutations are reason for many diseases and they can cause

fatal syndromes in children. [1] However, mitochondrial medicine is newly established

field and it is evolving very fast. Mitochondrial replacement therapies (MRT) are new

technology that allows women with mitochondrial disorders to give birth to healthy

children. Mitochondrial replacement therapy in oocytes or zygote could prevent

transmission of mitochondrial DNA defects to second generation and there are three

therapies: pronuclear transfer, mitochondrial spindle transfer and polar body transfer. All

three principles are using cytoplasm with mitochondria from healthy donor woman who

does not carry mitochondrial DNA mutation, separating the nucleus from mutated

mitochondrial DNA and putting that nucleus into cytoplasm with normal and healthy

mitochondrial DNA. However, this topic rises many ethical questions and number of

worries to what this scientific innovation will lead to. In this paper we will analyze

principles of mitochondrial therapies as well as all positive and negative sides, concerns

and questions that have been raised about mitochondrial replacement methods.

Unfortunately, there is no cure for these disorders caused by mitochondrial DNA

mutations and treatment is limited. Option to reduce the risk of transmitting

mitochondrial DNA mutations is put on future parents.

Key words: mitochondria, mitochondrial replacement therapy, mitochondrial

DNA,

techniques, ethics
Introduction

In health care, when approving a new drug or therapy, the critical step is to transit

new biotechnological innovations into clinical trials. Pharmaceutical companies as well

as the US Food and Drug Administration are facing serious challenges when these

clinical trials need to recruit volunteers in a right and ethical way. Two still developing

fields, reproductive medicine and genetic engineering, are trying to improve human

health in many ways thus allowing scientists to deal with the over 6,000 genetic

mutations that cause severe diseases that our population face today. The highlight of this

paper is new technology that rises from combo of these two fields, so called

mitochondrial replacement therapy. [2]

Try to imagine that there is regulation and law which prevent 150 children a year

suffering from liver or kidney failure from receiving transplant. Probably something like this

would be unethical. However, this is the case for around 150 children every year in the UK

suffering from mitochondrial disease. [2] Mitochondrial transfer is described as a form of

transplantation which means transfer of healthy mitochondria to a people with diseased

mitochondria. This procedure is done by taking the egg from healthy woman and removing

entire nuclear DNA. In this way healthy organelles as well as mitochondria are out of that

cell. After that, nuclear DNA is transferred from patient’s egg. [2] This combination looks

like organelle transplant but in this case child will be genetically the child of nuclear donor

just without mitochondrial disease. MRT could prevent transmission of mitochondrial DNA

(mtDNA) defects to offspring. Mitochondrial replacement is a special form of in vitro

fertilization in which the future baby's mitochondrial DNA comes from a third (healthy)

person. This is used in cases when mothers carry genes for mitochondrial diseases. It
involves removing eggs from a woman, removing sperm from a man, fertilizing the egg with

the sperm which allows the fertilized egg to form a blastocyst, and after that implanting
the blastocyst. MRT involves an additional egg from a third – healthy person, and

manipulating both the recipient - patient egg and the donor egg. [1]

Mitochondria and its role

In fertilization process, woman’s egg provides all cytoplasm for embryo. That is

the reason why mitochondrial diseases are only inherited from maternal line. [2] Sperm

do have mitochondria on their way to ovum but they are degraded through a process

ubiquitination which means proteolytic destruction of male mitochondria when sperm

fertilizes egg. [3] Mitochondria are very important and essential organelles. Most of cell’s

DNA in located in nucleus while mitochondria is located in cytoplasm and also contain

small fraction of organism’s DNA. It is called mitochondrial DNA(mtDNA) and it counts

for less than 0.1% of total amount of DNA contained in the cell. [3] Mitochondria contain

only 37 genes, and they are essential for energy supply of cells. However, defects in

mtDNA can cause devastating diseases as mitochondria are providing energy that cell

needs in order to function. [7]

Mitochondria inheritance patterns differ among individual but there are two main

transmission patterns: homoplasmy and heteroplasmy. If a mother is homoplasmic, she has a

single variant of mitochondrial DNA. All her offspring will inherit the identical mtDNA

because all her oocytes will have same mtDNA. [7] The other pattern is known as

heteroplasmy. It represents multiple variations in mitochondrial DNA which can cause

unequal distribution of healthy and mutant mtDNA. In that way, small number of
mitochondrial DNAs can be over-represented in children because the mitochondria is passed

down preferentially. [11] In heteroplasmy is important

to predict mother’s likelihood of transmitting mitochondrial disease to child. It may not

be so precise in clinical trials because mother may pass large but also small number of

mutant mtDNA

[1] However, heteroplasmy is not so bad since it can be that just small amounts of mutant

mitochondrial DNA is into resulting embryo.

Mitochondrial DNA differs from nuclear DNA in several ways. Firstly, mtDNAs

are present in multiple copies within single cell and this fact can affect way in which

mitochondrial disease is expressed. Sometimes it can affect all copies but on the other

hand it can also affect just small number. [1] According to this, individuals with defects

in mtDNa are affected according to number of affected mtDNA copies; patients can be

mildly or severely affected. For transmission of mutated mtDNA that potentially can

cause disease has been reported at a frequency of 1 in 200 in newborns. However, there is

no cure for such mutations and there are also just few available therapies but they can just

alleviate the symptoms of disease. [11]

Diseases transmitted through mitochondria

When we compare nuclear and mitochondrial genome we can see that

mitochondria, containing uncountable copies of the mitochondrial genome, has about

100-fold higher mutation rate than the nuclear genome. This fact makes mitochondrial

DNA heterogeneous which can be either within the same cell or mitochondrion, and that

is why mitochondrial is considered as heteroplasmic. During a cell division,

mitochondrion is being segregated between the two daughter cells in a random manner,

and this process is not well organized and precise as nuclear

chromosome segregation (mitosis). At the end of division, daughter cells get similar

copies of their mitochondrial DNA, but not identical. [11]

Moreover, patients who suffer mtDNA diseases have to wait for further treatment

advances since it is now only possible to treat early signs of this disease including

epilepsy, diabetes and cardiac diseases. [1]

Mitochondrial disease is not just single disease. This term is used for many

syndromes associated with mitochondrial mutations. Some of the syndromes are: Kearns

- Sayre syndrome and Leber Hereditary Optic Neuropathy (loss of central vision). Later,

other diseases such as ophthalmoplegia (weakness of the eye muscles, dropping eyelids),

Rhabdomyolysis (breakdown of muscle tissue that leads to the release of muscle fiber

contents into the blood, substances are harmful to the kidney and often cause kidney

damage) were associated with mitochondrial mutations.

There is some evidence that mitochondrial diseases are associated with some cases of

diabetes, hypertension. [3] According to the Foundation for Mitochondrial Medicine,

mitochondrial dysfunction has a role in many common diseases such as autism and

Alzheimer’s disease. [11]

According to the United Mitochondrial Disease Foundation, there are a lot of

symptoms in almost every organ system that could be associated with mitochondrial

disease. Below is a list of organs and potential symptoms of mitochondrial disease in

those systems:

• Brain - developmental delays, dementia, migraines, strokes

• Nerves - weakness, absent reflexes

• Muscles - weakness, muscle pain, gastrointestinal problems

• Liver - liver failure

• Ears and eyes - visual loss, hearing loss, strabismus
 • Systemic - respiratory problems, failure to gain weigh

Techniques for mitochondrial replacement

Patients diagnosed with mtDNA diseases show common symptoms like weakness

and fatigue, different seizures, delays in development, vision loss, cardiac problems, and

sometimes even premature death. The main reason for introducing MRT in science and

medicine is to prevemt the transmission of mitochondrial DNA diseases from mother to

offspring. This can be done by creating an embryo containing nuclear DNA from the

deliberated mother and mitochondrial DNA from a woman who does not have potential for

developing a mtDNA disease of any kind. [8]

Mitochondrial replacement technique works on principle that it uses healthy

mitochondria from egg donor-woman using 3 possible techniques. First one is called

maternal spindle transfer (MST). In this technique healthy nucleus of an egg with

affected mitochondria is removed and transferred to egg of donor which contain healthy

mitochondria. Second method is called pronuclear transfer (PNT). In this method transfer

happens after fertilization of oocyte. Two oocytes are fertilized with the father sperm.

After that, pronuclear which contain mother’s mitochondria is transferred to cell

containing healthy mitochondria. [7]

Pronuclear transfer (PNT)

In pronuclear transfer, an oocyte is removed from the patient and fertilized with

sperm from father. Then, donor oocyte is fertilized with sperm from father.
Zygote stage in mammals is characterized by the presence of two pronuclear (PN) and each

containing a haploid chromosomal number which is complement of nuclear DNA forms

sperm or
oocyte. [5]

Steps:

1. At day one of embryonic development, while the embryo is still a single undivided

cell, the two pronuclear, or unfused nuclei of the egg and sperm, are removed from

the cell for transfer. Removal of the two pronuclear leaves behind almost all of the

mother’s unhealthy nuclei. The cell without nuclei is then discarded.

2. Separately, a second embryo is created from the egg of an unrelated female donor,

which has healthy mitochondria, and the intended father’s sperm. The second embryo

is also enucleated at day one of development.

3. The intending parents’ pronuclear are then inserted into the second enucleated
embryo.

4. The new embryo now contains the pronuclear DNA of the intended parents and the

healthy mitochondria from the female donor’s egg.

5. The embryo can now be transferred back into the intended mother and can continue

to develop unaffected by inherited mitochondrial disease.

The male and female pronuclear are removed from each fertilized egg prior to their

fusing, and the pronuclear from the recipient's fertilized egg are inserted into the

fertilized egg from the donor. As with MST, a small amount of cytoplasm from the

donor egg may be transferred, and as with MST, the fertilized egg is allowed to form

a blastocyst, which can then be investigated with preimplantation genetic diagnosis to

check for mitochondrial mutations, prior to being implanted in the recipient's uterus.

[5]
Spindle transfer (ST)

Maternal Spindle Transfer is a technique similar to pronuclear transfer in a way

that the aim of technique is to prevent transmission of mitochondrial disease. However,

the main difference between these two techniques is that maternal spindle transfer uses

unfertilized eggs instead of the early embryos used in pronuclear transfer. [6]

In MST, an oocyte is removed from the recipient, and when it is in the metaphase

II stage of cell division, the spindle-chromosome complex is removed. In this process

some of cytoplasm comes with it, so some mitochondria are likely included. The

spindle-chromosome complex is inserted into a donor oocyte from which the nucleus

has already been removed. This egg is fertilized with sperm, and allowed to form a

blastocyst, which can then be investigated with preimplantation genetic diagnosis to

check for mitochondrial mutations, prior to being implanted in the recipient's uterus. [6]

Steps:

1. Intending mother’s egg is extracted from her ovaries. The cytoplasm of the intending

mother’s eggs contains the unhealthy mitochondria.

2. Chromosomes, the nuclear DNA material, are found in the intending mother’s eggs are

grouped together in a spindle-like formation. The chromosomes are removed for transfer

to the donor egg. The chromosome-free egg, which contains the unhealthy mitochondria,

is then discarded.

3. Separately, a donated egg is also extracted from an unrelated woman who has healthy

mitochondria. Similarly, the chromosomes of the donor’s egg are removed. However,

these chromosomes are discarded, leaving behind the healthy mitochondria in the

cytoplasm.
4. The spindle-like chromosomes previously taken from the intended mother’s egg are

inserted into the enucleated donor’s egg.

5. The resulting reconstructed egg contains nuclear DNA from the mother and the

healthy mitochondria from the donor.

6. The resulting egg can now be fertilized with sperm from the intended father. The

resulting embryo will be implanted into the intending mother and will develop

unaffected by inherited mitochondrial disease. [13]

Researchers at the Oregon Science and Health University announced that they had

successfully completed maternal spindle and three healthy offspring were produced

through the use of maternal spindle transfer. After birth, there was no mutated

mitochondria from mother. After this procedure, growth is monitored monthly and at the

age of two there were no differences from experimental controls. Currently, researchers

at Newcastle University in the United Kingdom are collaborating with the Oregon

researchers. The Newcastle group is testing the maternal spindle transfer technique on

human embryos. No results have been published. [6]

Polar body transfer (PBT)

During meiosis, oocyte undergoes two reductive divisions. In these divisions

there is uneven cytoplasmic segregation of two small bodies, called polar bodies. The

first polar body (PB1) contains a diploid set of chromosomes, and the second (PB2)

contains a haploid set.

In polar body transfer, a polar body (a small cell with very little cytoplasm that is

created when an egg cell divides) from the recipient is used in its entirety, instead of using
nuclear material extracted from the recipient's normal egg. This can be used in either MST

or PNT. This technique

was first published in 2014 and as of 2015 it had not been consistently replicated, but is

considered promising as there is a greatly reduced chance for transmitting mitochondria

from the recipient because polar bodies contain very few mitochondria, and it does not

involve extracting material from the recipient's egg. [1]

Society and culture view

Regulation

In the year 2015, United Kingdom’s regulations on mitochondrial donation came

into force and became the first country to legalize the procedure. This allowed IVF

centers to include mitochondrial donation techniques in in vitro fertilization treatments.

The idea was recommended in 2013 but regulatory authority published regulations in

2016.

On the other hand, USA, till February 2016, had no regulations for mitochondrial

donation and any application that involved implanting embryos into a woman was not

allowed. The application and approval of MRT falls under FDA, and for it to be accepted

clinical trials are needed, considered only under investigation of new drug application.

Unlike the in UK, there is no specialized agency in USA that is in charge of dealing with

assisted reproduction. USA, in the 1990s, had negative experience with cytoplasmic

transfer which influences the area of mitochondrial transfer today. This is what made

current regulatory landscape of MRT in USA very unpredicted and unstable. The failed

technology was invented for women having implantation failure. It was halted after the

birth of two children with different genetic disorders even though it also helped many
families to have their long desired children. The exact number of born children is not

known and they have been lost in means of following them up. [10]
Ethics

Even with promising outcomes of mitochondrial replacement techniques, this

topic raises many ethical and social questions since it involves modification of germline

and such modifications would be passed on offspring’s.

Another controversial thing is using embryos for in vitro research, as embryos are created

for research and the financial compensation of egg donors. [11]

Question that rises debates was weather genetic identity of children born as a result of

mitochondrial replacement affect their emotional well-being if they are aware that they

are different from others children from two parents. [13]

There is of course people who argue that scientists are "playing God" and that children

with three genetic parents may suffer from psychologically and physically. [11]

In February 2016, Institute of Medicine of the National Academies of Sciences,

Engineering and Medicine publish a report weather clinical research into mitochondrial

replacement is allowed and permissible.

The report concludes that it is 'ethically permissible' to continue clinical investigations of

MRT, until certain conditions are met. They recommended that initially it should only be

used for male embryos to ensure that DNA with mitochondrial disease won't be passed

on. [4]

Third parents’ rights and responsibilities

As well as sperm or egg donor, mitochondrial donor would donate their germ

cells to bank. After that it would pass screening then hormone administration and at the

end procedure of extraction of gamete cells. [11]

In United Stated, as a sperm donor you have no right or responsibilities to your sperm

after donation. Licensed clinics are required to have strict legal, ethical and medical

standards. [11]

As well as sperm donors, mitochondrial donors are also following this model.

They have no ties to the child that they helped to conceive. They would just simply make

their donation anonymously and never look back.

All characteristics such as organ structure and function, intellectual and emotional

characteristics are derived from nuclear donor. When person receives a heart transplant,

nothing of moral importance changes, it is same with mitochondrial replacement. [13]

Designer babies?

“Designer babies” refer to potential babies whose traits have been manipulated

using technology. [11] People believes that mitochondrial replacement could lead to

human experimentation.

As we have already said, mitochondria have their own DNA, and that means that

they are responsible for certain traits. Replacing the mitochondria of embryos means that

you are essentially changing their genetic material. This leads to debates such as designer

babies and of course genetic manipulation. It will be very hard to control genetic

manipulation if creating designer babies is allowed. [11]

However, there are people who think otherwise. They believe that mitochondrial

replacement is the best possible cure for inheritable mitochondrial disease. In their

opinion this procedure will be done to cure potentially fatal diseases not to choose child’s

eye and hair color.

 Fertility experts thought that mitochondrial disease is not a case of designer

babies and that media is responsible for presenting it to the other people as designer

babies problem. [10]

Limitation of technique

In mitochondrial replacement, all subsequent germ lines of that cell are altered as

well which means that embryo’s children and all future generations will inherit

transplanted mitochondrial DNA. [11]

However, even if mitochondrial replacement is meant to cure disease, there is

always a risk of uncertainty which is very alarming when person’s genetic traits are

manipulated and will be passed on to the rest of the germline. You can no go back if

something goes wrong. [8]

There is believe that mitochondria actually have an intimate relationship with

nuclear DNA and can have strong influence on phenotype. [9]

One study revealed that mitochondrial DNA is significant to biological metabolism.

Researchers concluded that cytoplasmic genes affect phenotype in equal amount as

individual nuclear genes because cytoplasmic genes are involved in production of

metabolite. [9]

Mitochondrial replacement is becoming more questionable if we consider that

mitochondria is much more than just battery power of cell and that it can actually affect

phenotype.

Conclusion

Mitochondrial medicine is newly established field which is rapidly evolving.

Since 1988, when mutations in mitochondrial DNA were discover to cause disease, more

than 100 clinical disorders have been recognized as mitochondrial disease. [13]
All three methods that have been described are using cytoplasm with mitochondria from

donor woman who does not carry mitochondrial DNA mutation. They are based on

principle of
separating the nucleus from mutated mitochondrial DNA and putting that nucleus into

cytoplasm with normal and healthy mitochondrial DNA.

However mitochondrial transfer is sometimes misleadingly described and

represented as three persons in vitro fertilization. Three people are not having child

together and child does not have mix of characteristics of three people. Mitochondrial

replacement therapy is similar to any other organ transplant, where recipient of organ

does not have any characteristics of organ donor. In mitochondrial replacement, nucleus

of the cell with DNA is not affected and that means it is not consider as genetic

engineering that could be used to create designer babies.

Doing mitochondrial DNA replacement at very early stage of embryo

development, disease could be cured and also children of the offspring will be free from

mitochondrial disease as well. This lead to complete eradication of disease from family.
References:
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May 20, 2018. https://www.nature.com/articles/nature20592.

[2]Savulescu, Julian. "Mitochondrial Disease Kills 150 Children a Year. A Micro-

transplant Can Cure It." The Guardian. February 02, 2015. Accessed May 20, 2018.

https://www.theguardian.com/science/2015/feb/02/mitochondrial-transfer-micro-

transplant-parliamentary-debate.

[3]Craven, L., and H. A. Tuppen. "Pronuclear Transfer in Human Embryos to Prevent

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Accessed May 20, 2018. https://www.ncbi.nlm.nih.gov/pubmed/20393463.

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