Therapy of Evil
How technologies for the treatment of mitochondrial diseases are going to be legalized for 24 years
Polina Loseva, N+1
For links, see the original article
We could have treated some genetic diseases 20 years ago by mitochondrial transplantation. But real attempts to apply it ended with accusations of scientists in eugenics, scandals and prohibitions, and mitochondrial diseases remained incurable. Since then, biotechnology has stepped forward, we have genome editing systems and the first patients whose genes have been rewritten. It's time to make another approach to mitochondrial genes. Will we be able to do without scandals this time?
First steps
When, in August 1996, doctors from a clinic in New Jersey injected Mr. Ott's sperm into 14 eggs of Mrs. Ott, no one yet knew which one of them would turn into little Emma and how this story would end for patients with mitochondrial diseases. Then the Ott couple was ready to take any risks after 6.5 years of futile attempts to conceive a child, and Dr. Jacques Cohen hoped for the success of his new technique. Its essence was simple: in the process of artificial insemination, doctors injected not only the sperm of the father into the mother's egg, but also a tenth of the cytoplasm from the egg of a young donor woman.
Of the 14 eggs fertilized in this way, six began to develop normally, four were planted in the mother's body, one took root, grew into Emma Ott and was born on time without complications. Cohen and colleagues reported in The Lancet magazine that they successfully managed to restore the fertility of a 39-year-old woman whose previous embryos developed incorrectly. New York newspapers advertised their successes with might and main. Dozens of infertile couples turned to the clinic for help, and over the next four years, 16 more confirmations that the technique works were born.
And then the thunder came.
Cohen and colleagues continued to improve their methodology and monitor the results. In 2000, they discovered that traces of donor genes remained in various germ tissues and cells of newborns who were born as a result of cytoplasm transplantation.
Embryos on the third day after fertilization. (a) mother's egg + father's sperm; (b) mother's egg + donor's sperm; (c) donor egg + father's sperm, (d) mother's egg + father's sperm and donor ooplasm injection (Carol Brenner et al. / Fertility and Sterility, 2000).
Perhaps this observation would have gone unnoticed if in 2001 they had not published another short report on long-term observations of children. This time they found traces of donor genes in the blood and cheek mucosa of two one—year-old babies and honestly announced: "this is the first case of inherited genetic modification" - which ruined the whole thing.
The phrase continued with the words "... which led to the birth of normal healthy children," but no one cared anymore.
The media rushed to discuss the "world's first GM children" and talk about the return of eugenics. The FDA, the American equivalent of Roszdravnadzor, required reproductive clinics to consider the use of donor eggs an experimental procedure and obtain special permission for them. By erecting a "paper wall", the bureaucracy curbed the technology and actually buried the controversial method.
A stranger inside
Cohen himself did not set out to create genetically modified people and did not even recognize his method as a modification — all the genes of the child remained in place and did not change in any way. He simply believed that the cause of infertility lies in the aged eggs of women and was looking for a way to rejuvenate them. Moreover, the doctors from his team made sure that no foreign chromosomes got into the microcapillary (with which sperm and donor cytoplasm were injected into the egg) — they only needed cytoplasm from the donor, and it was taken from the side of the egg where there was no genetic material. They coped with this part of the procedure successfully: no foreign nuclear genes were subsequently found in the children's blood.
Injection of a sperm into an egg (CC0).
However, along with the donor cytoplasm, other parts of the egg, including mitochondria, could get into the embryo. By themselves, they can even be useful: additional mitochondria can provide the developing egg with additional energy.
Mitochondria have their own genome inside. It was him that Cohen found in the cells of children, which prompted him to use the phrase "genetic modification" that so frightened the decent public.
Heteroplasmia — the neighborhood of several types of mitochondria in one cell — does not in itself affect intracellular life. Moreover, it naturally appears in aging human cells as mitochondria accumulate mutations. Therefore, there is no reason to think that someone else's mitochondrial DNA could affect the fate and development of children. In 2016, Cohen and colleagues reported on the health of already grown-up "experiments": no serious developmental abnormalities, no serious illnesses, good grades at school.
(a) Eggs 10 minutes after injection of donor ooplasm (red) (b) Trypronuclear zygotes 24 hours after injection of donor ooplasm. According to scientists, the red dots are precisely mitochondria (Jason A. Barritt et al. / Human Reproduction, 2001).
But the scientific community was not only concerned about the health of children. A much more important argument was the fact that some of these children — including Cohen's "firstborn" Emma Ott — are girls, which means they can pass on their unusual mitochondrial composition by inheritance, giving rise to a clan of "unnaturally" heteroplasmic people.
Since then, there has been evidence that heteroplasm in cell cultures can be reversible, and alien mitochondria in a foreign land are gradually dying out. But many participants in Cohen's research refused to test the blood of their adult children for heteroplasmia, and we hardly know now how well-founded the FDA's concerns were. The regulator's ban remains in force to this day, and scientists had to look for workarounds to infertility treatment.
Second mother
Cohen was never able to say for sure which part of the donor cytoplasm, if not rejuvenated the eggs, then at least helped women get pregnant. These could be not only organelles, but also some individual molecules from the young cytoplasm, for example, proteins or informational RNAs. Nevertheless, the scientist's work has created an important precedent: a third person's donor material can be used to create a child. And as soon as his experiments stalled under the scrutiny of the FDA, further progress moved to China.
Shortly after the FDA changed the rules of the game, Cohen's competitors moved their experiments from New York to Guangzhou, where no bans yet existed. There, a young embryologist John Zhang came up with the idea to do the opposite: if you can transplant a section of cytoplasm from a young egg into an old one, then why not try to do the opposite — transplant the nucleus of an old egg into a young one? Nuclear transfer technology (later called pronucleus transfer) he tested it in 2003: he fertilized the old (maternal) and young (donor) eggs, then removed the nucleus from the second one and transplanted the nucleus of the first one there.
(a) Spindle transfer (Zhang's Mexican experiment) (b) Pronucleus transfer (Zhang's Chinese experiment). Steve Connor / Nature, 2017.
It is difficult to say how successful the experiment was. Five embryos began to develop in the culture at once, which were transferred to the patient. Three of them took root at once. Scientists decided that it was dangerous, and caused the abortion of one of the embryos, and the other two later died themselves. Therefore, Zhang, unlike Cohen, could not prove that his technique is safe. Experiments were banned again — this time by Chinese regulatory authorities, citing the suspicious proximity of research to attempts to clone a person (but it is prohibited in China).
But the story, of course, did not end there: this controversial infertility therapy (pronucleus transfer) continues to be used even now. In 2016, it began to be used in Ukraine, in 2019 the first such child appeared in Greece.
Change of course
Those who did not believe that mitochondria could "rejuvenate" the egg, outlined another potential exhaust from this method. The transfer of pronuclei could be a way to get rid of mutations in mitochondrial genes. Quite often, such mutations make their carriers disabled at an early age: since mitochondria supply energy to cells, its main consumers — muscles and nerves - suffer most often. The carrier of such mutations cannot conceive healthy children naturally, since the father cannot help with mitochondria in any way: their child inherits strictly from the mother.
Thus, the transfer of pronuclei could be used as a therapy for mitochondrial diseases. Several research groups drew attention to this at once. Russian-born American biologist Shukhrat Mitalipov, known as a pioneer of human genome editing, founded the company Mitogenome therapeutics back in 2013 and began testing the technique on macaques. Professor Mary Herbert from Newcastle, UK, obtained permission to carry out the first such procedure in 2017. But John Zhang, having suffered a fiasco in China with the repair of infertility, still managed to be the fastest.
John Zhang with his first child from three parents (New Hope Fertility Center).
The first "his" child was born in Mexico in 2016, where the authorities are not so concerned about the regulation of childbirth. The boy's parents were Muslims, and the classical method of transferring pronuclei was impossible for them — for this it would have been necessary to destroy the fertilized egg of the donor, that is, to kill the embryo, which the religious norms of the parents did not allow. Therefore, Zhang used an alternative method — spindle transfer, that is, he first transplanted the mother's genetic material into a donor egg (without a nucleus), and then arranged a "date" with her father's sperm. But even such a trick did not appeal to the world community. The born boy was dubbed "the child of three parents," and a new scandal began.
The doors are closing
Some scientists accused Zhang of experimenting on living people, while others suggested conducting similar tests only on male embryos, which obviously would not transmit the "result" of the experiment to offspring. Still others asked the question: does Zhang have evidence that the child will not have heteroplasmia or even a rollback to the original state? Zhang had no evidence: the parents took the child and refused long-term supervision.
The outcome of the scandal was predictable: the FDA strengthened the previously erected "paper wall" and banned any manipulation of mitochondrial replacement. The UK remains the only country where they are now officially approved — in rare cases and after long discussions at the top, in the offices of the Human Fertilization and Embryology Department. Everyone else who wants to experiment with eggs and their mitochondria has to look for a country where the legislation does not regulate this technique in any way, and not to advertise their research too much.
Mitochondrial diseases could be the first genetic diseases that people have learned to treat en masse — but did not. The name "child from three parents" has firmly stuck to the mitochondrial transfer technique, and despite the fact that the researchers themselves consider it incorrect — there are only 37 donor genes, and there are 20 thousand of them from the father and mother — it is now steadily associated with a violation of ethical norms. Therefore, in order to solve the problem of infertility or to save your child from the risk of becoming the owner of a whole bunch of incurable diseases, parents have to go on "embryological tours", sometimes to the other side of the world.
An EM snapshot of the mitochondria. The black dots close to the membrane surface are mtDNA labeled with gold particles (Francisco J Iborra et al. / BMC Biology, 2004 / CC BY 2.0).
And then there was a way to cure genetic diseases hidden no longer in the cell's organelles, but right in its nucleus. Despite the fact that the people who first came up with the idea of applying CRISPR/Cas9 to human genes warned in advance that the system was not ready for this yet, history repeated itself. Taking advantage of the fact that Chinese legislation closed the gate for manipulating mitochondria, but said nothing about gene editing, another pioneer Jiankui He tested CRISPR on embryos. Then the same thing happened as always: a scandal, bans, attempts to prevent a repeat of the situation with "children from three parents" (however, WHO has been working on standards for the supervision of manipulations with the human genome for a little over a year now, and stubbornly avoids the word "moratorium"; meanwhile, in many countries, an official ban on CRISPR-there are still no children).
But since it is still necessary to treat genetic diseases, a compromise option has appeared - CRISPR therapy. In other words, while the world is figuring out whether we have the right to edit embryos, we can train on adults: inject an editing system into their blood and repair breakdowns directly in working tissues. This method has already been worked out on a variety of cells, and recently switched to in vivo testing.
As CRISPR conquered one therapeutic area after another, it became clear that it was powerless against mitochondrial mutations. The fact is that most genetic editing systems work like scissors, cutting the DNA in the agreed place. And if the nuclear DNA after such a cell easily restores, connecting the ends of the gap, then it destroys the mitochondrial one — normally it is rolled into a ring, so that a double-strand break is considered not an ordinary breakdown, but a sign of a serious problem. Therefore, the losses from such editing may exceed the winnings.
So mitochondrial diseases not only did not become the first achievement of genetic therapy, but also remained the last bastion not taken at all.
Parallel roads
In fairness, it should be said that embryo modification is not the only way to cope with mitochondrial defects. For example, mitochondria can be transplanted not into an egg, but into an already born organism (just as CRISPR/Cas is being injected now).
Two therapies of this kind are currently undergoing clinical trials. As part of the first — mitochondrial augmentation therapy — the child receives donor mitochondria from the mother (in case his mitochondrial disease originated from scratch, and did not get from the mother). Cells are taken from the child — for example, blood stem cells — and cultured together with mitochondria isolated from the mother's cells. It is believed that in this case, the child's blood cells absorb the maternal organelles, become more viable and will actively multiply after returning to the body, thus supporting the work of the "broken".
The second therapy assumes that the child becomes a mitochondrial donor for himself — for example, in the case of cardiac ischemia during childbirth or in the first hours of life. Then a piece of tissue is cut out of some skeletal muscle, mitochondria are isolated from there and injected into the heart muscle. This method was recently tested on five newborns: two of them could not be saved, and three more recovered, but it is unknown what role mitochondrial autotransplantation played in this.
One can imagine that a combination of these two methods could give rise to a full-fledged therapy, during which donor mitochondria would be injected into the patient's blood, and they would populate the tissues damaged by mitochondrial disease. However, the scientific community still has many questions about these procedures. Despite the fact that individual mitochondria can indeed survive in blood plasma, it is unknown whether the body cells are able to capture them, and if so, whether they survive inside. Defenders of the method note that "sometimes it is necessary to adopt the technology, even if we do not know how it works."
There are also more radical solutions to mitochondrial problems: for example, a few years ago, the most famous fighter against aging, Aubrey de Grey, proposed transferring all genes from the mitochondria to the nucleus. His colleagues managed to move two of them and show that even from there they successfully coped with their duties.
And although this project seems even less realistic than all the others, it may turn out that some mitochondrial genes can be transplanted separately — just as they try to cope with mutations in nuclear DNA with the help of gene therapy. There are also such works, there are also the first clinical trials — this is how they try to treat hereditary optical neuropathy. The trick here is that gene therapy delivers the mitochondrial gene not to the mitochondria, but to the nucleus. Nevertheless, it is possible to construct an artificial gene so that the resulting product is transported by the cell to the mitochondria, and then it does not matter where it is produced.
A new trail
And yet it would be much more reliable to rewrite the mutant mitochondrial gene once and for all. This task was taken up by David Liu, one of the world's top genome editing specialists. It was he who in 2016 came up with how to correct mutations without cutting DNA — and assembled the base editor. This is a molecular system of two enzymes: dCas9, which is induced to a specific place in DNA, and deaminase, which corrects one nucleotide to another, literally rewriting the "genetic text" for profit.
This method is not suitable for mitochondria either. Base editors directly depend on the guide RNA that delivers them to the target: then Cas unwinds the DNA helix into two separate strands, RNA binds to one, and deaminase attacks the other. But the guiding RNA cannot penetrate into the mitochondria — there is not enough transport system that would drag it through two membranes. It was necessary to come up with some kind of system that works without RNA. Recently, Liu's team created such a system. And it also works without Cas.
The system is based on the DddA antibiotic, which is secreted by the bacterium Burkholderia cenocepacia. It has two important features: firstly, it acts pointwise: it corrects all C (cytosine nucleotides) to A (adenine) in the target gene - more precisely, first, it converts C to U (uracil), and the cell turns them into A — that is, it works by deaminase. Secondly, unlike all other base editors, it binds to double-stranded DNA — which means there is no need to divide it into two strands using a guide RNA that does not fit into the mitochondria.
But just like that, you can't do without a guide RNA anyway — some other mechanism is needed to target DddA to the right place in the mitochondrial genome. And here, Liu's team took a step back and used technology that, it would seem, had long since given way to CRISPR — TALEN. These are bacterial enzyme constructors: they are built from domains, each of which recognizes a specific DNA sequence. By selecting the right set of domains, it is possible to ensure that the enzyme sits on a specific place in the genome. This technology, which has long been considered more complex and expensive, can now close the niche that CRISPR was too tough for.
By combining the selected TALEN with the non-toxic part of DddA (the one that can only deaminate DNA, and not recognize its sections), Liu's team received the coveted tool. However, it is still too damp for clinical use: in various experiments, he was able to rewrite no more than half of his targets in cells. Nevertheless, it penetrates into the mitochondria and does not destroy them from the inside, and this is much more important than efficiency, which is easy to increase.
And if this can be done, then we can assume that there is no longer such a gene in the human body that we cannot change. There will not be a single piece of DNA that will be beyond our control.
Liu's tool does not require any "third parent", and his work does not even remotely resemble cloning. This means that he has a higher chance of not hurting anyone's feelings and not causing justified fears. But what will be the next twist of this plot? There are two options: long and thorough trials and gradual application of the new editor on adults (as happens, for example, with gene therapy) or an adventure involving embryos and attempts by another pioneer to get ahead of his time (this was the case with mitochondrial transplantation, it was the case with CRISPR and, perhaps, it will be more than once). The mitochondrial editor is still a long way from the clinic. But it is already possible to bet on what fate awaits him in a few years: will it be another scandal, a ban and the search for a new road — or will it finally be possible to put a dot instead of a question mark at the end of this story?
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