In edit mode
Experiments on creating CRISPR children through the eyes of opponents and patients
Polina Loseva, N+1
Sergey Kutsev, director of the Medical and Genetic Research Center (MGNC), said in mid-September that Russian geneticists intend to require the Ministry of Health to impose a moratorium on the clinical use of editing human embryos. This is how he and his colleagues reacted to the statement of biologist Denis Rebrikov that the first pair of parents had already entered his experiment on the treatment of hereditary deafness. The editors of N+1 tried to find out how this experiment works from the inside. We talked with his participant (who has already decided to postpone the birth of a child during this time), with Rebrikov himself and his opponents.
To speak or to be silent
In November 2018, Chinese scientist Jiankui He said that during his experiment, two girls with an altered genome were born. According to He, the twins Lulu and Nana were supposed to become resistant to HIV infection.
Whether this was achieved, we still do not know – He did not publish his data either in a peer-reviewed journal or on the open website of bioRxiv preprints. But we do know exactly what the Chinese experimenter did wrong – from the moment of the first statement, a mountain of criticism fell on him, and some details that he could not take into account when planning a study still come up.
Denis Rebrikov, a Russian biologist, vice-rector of the Pirogov Russian National Research Medical University (RNIMU), head of the genome editing laboratory at the Kulakov Scientific Center, set himself a similar goal.
By the time of He's scandalous statement, Rebrikov already had one article on the genetic editing of embryos on his account. His research team also worked with the CCR5 gene associated with HIV resistance, but used a slightly different editing technique and managed to test it only on abnormal embryos – "by-products" of IVF technology, which, due to genetic defects, cannot be transferred to the uterus for women.
Despite the criticism that his Chinese colleague was subjected to, Rebrikov soon said in an interview with Republic that his group also plans to seek the birth of genetically modified HIV-resistant children in Russia.
Six months later, Rebrikov confirmed his intentions in an interview with the journals Nature and Science, and a month later – in July 2019 – unexpectedly changed his goal.
The new target for editing was to be the GJB2 gene, which causes hereditary deafness. In July, Rebrikov said that he already has several families who could take part in an experiment on editing embryos.
At this stage, Rebrikov was ready for a broad discussion of his plans. "We are public in order not to take a bad step at some point that no one will like," he shared his thoughts with N+1. "If we are banned from this experiment, then we will need to explain why, and this will create a situation of discussion that can be useful for science as a whole".
And the discussion began. On September 5, Rebrikov informed N+1 that the first pair of parents had already entered the embryo editing project. On September 17, the director of the MGNC, Sergey Kutsev, said that the Center intends to resist these experiments and is going to ask the Ministry of Health to impose a moratorium on them.
It is at this point that the paths of Denis Rebrikov and Jiankui He diverge. A Chinese biologist has confronted the scientific community with the fact: GM-children are born, information about them and their parents is classified. The scientist himself has not communicated with the press for several months, so the course of his research has to be restored for indirect evidence.
Rebrikov did not avoid publicity, and now we can discuss how his experiment works, even before it bears its first fruits. N+1 talked to Rebrikov's opponents from the MGNC and with his first, now failed patient, to find out whether the experiment withstands the criticism that Jiankui He was subjected to.
The disease is one or the wrong
The first complaint about the Chinese experiment is the choice of a target. He's team worked with the CCR5 gene, a mutation in which can make human cells immune to HIV, more precisely, to the most common variant of this virus. For his experiment, according to He, he found a married couple in which the father was HIV-positive and the mother was HIV–negative.
However, in this situation, editing would make sense only if the mother herself was ill and if she refused surrogacy. Otherwise, it would be enough to carry out simple procedures to clean the father's sperm from viral particles and the child would be born healthy.
In addition, HIV is not a genetic disease, it can be prevented by other methods. But even among truly genetic diseases, it is not easy to find a target that would justify the use of a new risky technology.
"It is impossible to imagine such a situation," says Mikhail Skoblov, head of the Laboratory of Functional Genomics at MGNC, "in which it would be impossible to help a family wishing to have a healthy child. Either choose the right embryo, or [exercise] control in the early stages of embryogenesis. That's why [preimplantation diagnostics] technologies have appeared, and they work perfectly, they solve all these problems that people come to our center with."
Rebrikov, however, managed to pick up a case where genetic diagnosis is not able to cope with the problem. His new target is hereditary deafness, or rather, the 35delG mutation in the GJB2 gene.
This gene is responsible for the formation of channels between neurons; in its absence, sensitive cells in the inner ear cannot generate a signal in response to sound, and a person loses hearing. Homozygotes for this mutation are usually deaf from birth.
The first participant of the experiment, Rebrikova Alina, was born almost deaf (name changed). "I was a long–awaited and beloved baby," she says. "However, after a while it turned out that I did not respond to sounds, and at the age of two my parents found out that I had hearing loss of 4 degrees, borderline with deafness."
Alina lives with a hearing aid and freely communicates with people, but she did not learn to speak right away. "I had a "wooden" language, so it was very difficult to put sounds mechanically: I learned to speak the letter "r" for four months, and the letter "k" for exactly a year. In order for classes with a speech pathologist to bring some effect, I lived in a special correctional boarding kindergarten for five days."
Alina's husband was born hearing, but in adolescence he gradually began to go deaf. Cases when a mutation in GJB2 does not manifest itself from birth are quite rare – and this is one of them. Now the man wears a hearing aid and is quite socialized.
A few years ago, he and Alina had a daughter. The girl also suffers from deafness. "We found out that my husband and I are homozygotes," says Alina, "and editing the gene at the zygote level is the only option for us to give birth to a hearing baby."
Skoblov, however, considers this target insufficiently justified: "That both parents are deaf, they both have homozygous mutations, and only this option can interfere here, it's true," he admits. "But hearing loss is not the kind of sore that requires such a story, not at all."
According to Skoblov, hearing loss is often found in the population precisely because it does not prevent patients from living and creating families: "These are people who live in their own society, they usually choose their own kind so that similar children are born, it is psychologically easy for them to communicate and exist with them."
Of course, there are still about 7 thousand hereditary diseases, carriers of which rarely live to adulthood, and methods for their prevention could be very much in demand. However, Skoblov emphasizes, it is for such cases that modern preimplantation diagnostic methods have been developed that will prevent the birth of children with genetic defects.
Rebrikov himself does not believe that genetic editing makes sense to work on really severe genetic diseases. "There are no such situations in nature," he said in an interview with the portal "Attic". – These people do not live to puberty, they do not reproduce. And situations [in which genetic editing is possible] all are soft. This is what people live with and create families."
As for deafness, Rebrikov refuses to consider this disease insignificant and recalls that for complete rehabilitation a child needs a cochlear implant, and this is actually the introduction of the device into the brain. "A hearing person, perhaps, should not have reasoned whether this is a sore or not, so as not to wish it to his children," he concludes.
A cochlear implant is a device that generates sound anew – unlike a hearing aid, which simply amplifies an external signal. The external part of the device – the receiver – collects audio signals from the outside and transmits them to the internal part – the transmitter. And he, in turn, sends electrical impulses to sensitive neurons in the cochlea. Thus, the cochlear implant actually performs the functions of the inner ear and allows you to compensate for the impaired sensitivity of neurons.
Location of the cochlear implant in the ear
Unlike her husband and daughter, hearing aids no longer help Alina, and her hearing is constantly falling. Now the woman is in line for cochlear implantation. "Like all operations, it has risks," says Alina. "But in my case, this is the only way to hear better."
Chicken or egg
The second claim to He's experiments is the use of "raw" technology. Most scientists have no doubt that it will one day become part of medicine.
"We need to move-develop [technology. – Approx. N+1]?" asks Mikhail Skoblov. And immediately answers: "Of course you need to. Is she the future? Of course, she has huge opportunities." However, scientists believe that its time has not yet come. "The whole world knows how imperfect the technology is today," Skoblov is indignant.
Today, the whole world knows that Jianku He failed to achieve effective genome editing in the first GM girls. Judging by the slides that the biologist demonstrated at the Hong Kong summit, in one girl the changes affected only one of the two copies of the CCR5 gene, and in the second – both, but in none of the cases it was possible to create the required CCR5Δ32 mutation, that is, to remove a specific sequence of 32 nucleotides from DNA.
Rebrikov believes that his technique works effectively. At least, in his article, which is devoted to working out the technique on abnormal embryos, it is said about 50 percent of embryos whose DNA is corrected with 100 percent efficiency.
There is still the issue of security. The CRISPR/Cas9 molecular system that He used, and now Rebrikov is going to use after him, should not only edit all copies of the gene, but also not affect other parts of the DNA (this is called off-target activity).
"No scientific group in the world," the MGNC press release quotes the words of Sergey Kutsev, "has presented evidence of the possibility of avoiding the so–called "off target editing" when neighboring genes are "touched", mutations in which are not detected." The consequences of such inappropriate editing can be very different. Kutsev, for example, mentions works that talk about the transformation of an accidentally touched gene into an oncogene.
Rebrikov has his own verification method: he sequences the genomes of the parents and the embryo after editing, compares them and looks for changes that could resemble manifestations of off-target CRISPR/Cas9 activity.
Skoblov believes that this is not enough: "Many things are not determined by genome sequencing. The genome is a very large and complex object, and the effectiveness of diagnosis and detection of various kinds of mutations is very low today. Firstly, a lot of different things can happen that Rebrikov simply won't see – related to epigenetics, first of all. It will not determine how the expression of different genes will eventually change. Secondly, genome-wide sequencing is not good at detecting all sorts of large permutations."
In response, Rebrikov recalls that mutations can occur in the genome of the embryo and de novo, that is, by themselves. Therefore, the purpose of the test, according to him, "should not be to prove the absolute absence of the so-called "off target editing", but to assess the degree of risk and compare this risk with the risk of new natural mutations occurring in large numbers during the "creation" of each of us." And according to the scientist, his method is able to cope with this task.
The only argument that could convince opponents of editing is the living result of the application of the technique, an organism without the side effects of interference with the genome.
"[It is necessary] to observe a generation, another. Now any GMOs – herring, potatoes, do so. Take the mice," Skoblov suggests, "try, repeat, show that everything is cool, everything works. Monkeys, dogs, whatever. Show the safety of this technology. Not theoretical, but real, experimental. You can't experiment on people."
Rebrikov also claims that experiments on animals will not give anything new now: "On animals we can only see an "average hospital" off-target effect for nucleases (enzymes that make breaks in DNA. – Approx. N+1) of this type. But this does not characterize the work of the system in the human genome."
"The only correct way to prove the safety of the drug," continues Rebrikov, "is to check the inappropriate activity of the system on the zygotes of a particular pair. But since the DNA of different people differs by 0.1 percent, demonstrating safety on one genome does not guarantee that there will be no problems on the other. Each time it is necessary to check on the genome of specific patients. Checking for somatic (not sexual. – Approx. N+1) cells of patients will also not give us an accurate answer to the question of off-target activity. I am ready to justify at any scientific conference why this position is the only correct one for today."
The current situation resembles the well–known chicken and egg paradox: what should happen first - an experiment on humans or an assessment of its safety? Skoblov and his colleagues from the MGNC would prefer the second option: "The technology must first be evaluated," he says, "what risks it has, what errors are possible, what real parameters. But she does not have these assessments, because they were not conducted."
Rebrikov believes that experiments are always the first: "It should be taken into account," he recalls, "that no new medical technology before its introduction into practice could guarantee the absence of unforeseen complications. If we proceed from these positions, we need to stop all innovative medical developments and just live in harmony with nature."
Immoral or not
The third accusation that the scientific community has brought against Jianku He is bias in talking to patients. He said that he personally communicated with the future parents of GM children and received informed consent from them. However, since the scientist was interested in patients entering his experiment, he could keep silent about the possible risks.
Whether this is true or not, we still do not know: in the document signed by the participants of the He project, it only appears that this is an "experimental treatment of HIV infection", and the scientist explained the details of the technology to them in words.
Rebrikov began negotiations with his patients more than six months after the Chinese scandal. Alina doesn't know much about He's experiments and doesn't seem to see any reason to worry about them.
"I've read about them, but only superficially, so I can't say anything significant. I can say that the same “enzyme” was used there, which should potentially correct one letter in the DNA, after which the baby is born hearing. Only such an enzyme was used against HIV, and, as I heard, the experiment was successful. I don't know anything else," she says.
A child with a cochlear implant
Alina found out about the new project through Instagram. Surdologists from the National Clinical Center of Otolaryngology wrote that they were looking for couples with hereditary hearing loss for free genetic testing, but did not mention any details.
In the comments, the authors of the post explained to interested readers that testing is the sequencing of an exome, that is, a "significant" part of the genome, there was no talk of other proposals.
The day after the publication of this post, Rebrikov announced that he "has candidates for editing." Alina and her husband decided to "check the genetics" because their first daughter is also deaf. They responded to the post, Rebrikov's phone was sent to them, they called and met at the end of August.
"At the first meeting," Alina recalls, "Rebrikov told us that we would have the opportunity to give birth to a healthy baby by selecting a healthy embryo, but on condition that one of us should have at least one allele without mutation. But if we turn out to be homozygotes for the 35delG mutation, then the only option for the birth of a hearing baby will be gene editing at the zygote level."
Unlike He, Rebrikov did not communicate with potential patients alone. "The conversation was not only with him, but also with the chief reproductologist of the Center of Obstetrics and Gynecology," Alina clarifies. – The conversation was very interesting and intense. My husband and I left under a great impression."
On September 17, the director of the MGNC called on the study participants to withdraw from the project. "These experiments are immoral," Kutsev is quoted as saying in the MGNC message. – I appeal to the couple who, according to media reports, agreed to participate in them: come to our Center, our doctors will explain to you at what stage the genomic editing experiments are at and what risk your family is at."
Apparently, his belated appeal was not heeded, but the patients themselves asked about the safety issue.
"The first question we asked him was about the risks,– says Alina. – We were interested in the question of whether there will be any risks with changing any mutations when the editing itself takes place. Denis honestly replied that there are always risks. But what exactly the consequences may be – we were not informed about this."
However, according to the woman, she did not sign consent to the procedure.
Will they forbid or allow
Jiankui He's experiments became possible only because at that time there was no law prohibiting them in China. A similar situation is developing in Russia. The MGNC report quotes the words of the deputy director of the Vera Izhevsk Center, according to which "strict legislative mechanisms for regulating research with editing of the human genome have not yet been developed in Russia."
It turns out that due to the fact that there is no direct ban on genetic modification, Rebrikov's experiments fall into a kind of "gray zone" - they are not prohibited, but they are not allowed either.
However, in order to conduct them, permission from the Ministry of Health will be required. On September 5, talking about the first couple of patients who entered the experiment, Rebrikov planned to apply to the Ministry of Health in a month. According to his calculations, it should have taken a week to get germ cells, another week for embryo cultivation, one and a half for genome sequencing and security checks, and after that the scientist expected to go for permission.
After this statement, Sergey Kutsev gathered a scientific council at the Medical and Genetic Center, at which the heads of the Center's laboratories decided to express their position. "The point is," Mikhail Skoblov explains, "that the professional community had to react somehow to the fact that it is wrong to do this, it is not good to do this."
According to Kutsev, in the near future scientists will prepare an open letter to the Ministry of Health and the Russian Academy of Sciences calling for a moratorium on the use of genetic engineering technologies on human embryos.
In previous conversations with N+1 Rebrikov looked to the future with optimism. "In general, the Ministry of Health is waiting for us," he said. Skoblov holds the opposite point of view: "We all believe that the Ministry of Health will definitely not approve of this."
To date, neither Rebrikov nor the MGNC scientists have published any applications, and the "race for the Ministry of Health" is just beginning. At first Rebrikov had a head start, but he managed to lose it – Alina left the experiment before reaching the stage of obtaining eggs.
"IVF and gene editing are methods," she explains. – The target is a child. We haven't had such a goal yet. We thought about the baby when we heard such an incredible opportunity about the birth of a healthy baby by ear. But after analyzing the whole situation with the family, we decided that we would postpone the birth of a child for a later period, because we are responsible not only for the upbringing, but also for the financial well-being of the family. If I go on maternity leave again, it will be extremely difficult to return to the working system. Besides, I'm on the waiting list for cochlear implantation. I don't think my company will provide me with so many sick days."
Alina says that Rebrikov is ready to wait and return to work with her later. But she is going to give him an answer only after the implantation operation. Whether the scientist has agreements with other married couples, she does not know.
Hype or reputation
Jiankui He was also accused of the fact that the main purpose of his experiment was fame, not helping people. "HIV is a very well–known disease," said a professor at the Kutafin Moscow State Law University (MGUA) Paul Kalinichenko. – Because heart defects or hemophilia are rare, they do not excite people so much, many have not heard of them at all. You can't create a sensation with them, but you can with HIV, it's a pandemic, a kind of tribune."
Denis Rebrikov, as expected, is reproached for the same thing.
Mikhail Skoblov believes that the biologist puts too much at stake for the sake of an unpredictable result. "I have a question: if he wants it so much and thinks that he will certainly become famous, then maybe he will step over some things? Will he identify any mutations [after editing] and decide for himself that they are not significant?"
Nevertheless, Skoblov admits that Rebrikov has taken a win-win position. Even if he is not allowed to experiment, the scientist argues, "he will wash his hands and say: Well, I've done everything, I'm good, but the Ministry of Health...".
Skoblov suggests imagining what would happen if Rebrikov were, for example, a physicist and said that he had come up with a miniature nuclear engine for home use, and then offered to build it at the institute. "Everyone abroad, as Russia, as scientists, will perceive us in this light," he is indignant, "that these things are not regulated in our country, that we treat them calmly."
Rebrikov is really calm about what is happening: "Each of the existing medical technologies looked dangerous and inapplicable at the start," he objects. – But people thought out an algorithm for putting it into practice – this is called translational medicine. We want to work out a coordinated application algorithm approved by researchers and the regulator, and move along it, performing all the necessary studies and checks in such cases."
To date, the statements of opponents do not add up to a dialogue. The public discussion that Rebrikov was counting on at the beginning of the journey is still based only on fragmentary statements. Supporters and opponents of genome editing talk about safety and the need for checks, but they don't seem to hear each other.
Well, the fate of technology and potential patients now depends on which of them will be the first to hear the Ministry of Health – which is still silent.
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