Revised edition
What can the world expect from the appearance of genetically modified humans
Alexandra Borisova, Daria Spasskaya, N+1
Chinese scientist Jiankui He announced on Monday that the first genetically modified people in history are already living among us: we are talking about the birth of twin girls who, with the help of CRISPR technology, have artificially altered the gene responsible for susceptibility to HIV. There is still a lot of uncertainty in this sensational story. First of all, the author announced the experiment itself not in the conventional way – by publishing it in a scientific journal, but in a video on YouTube. The university where Jiankui He worked disavowed this project, colleagues condemned the experimenter, and the Chinese authorities launched an investigation. N+1 asked scientists to tell how realistic the story told by the Chinese scientist looks, how accessible the method of genetic modification of human embryos is, what risks and dangers may arise in such experiments, why in most Western countries are prohibited from such experiments and can we expect genetically modified athletes, intellectuals or "service people" in the near future.
What happened?
In short: Jiankui He from the Southern University of Science and Technology in Shenzhen edited by CRISPR/Cas9 a zygote obtained as a result of fertilization of a mother's egg with a sperm of an HIV-infected father (with an undetectable level of viral load), modifying the CCR5 gene in it. This mutation makes a person less susceptible to the risk of HIV infection. Then the embryo was transplanted to the mother using standard methods used in in vitro fertilization (IVF), and as a result, twin girls were born - Lulu and Nana.
He states that another woman is now pregnant with a genetically modified baby, and seven other couples are participating in the experiment, which, however, is currently suspended "due to the current situation."
What is CRISPR-Cas
The history of CRISPR-Cas systems is far from human – in nature they are found in organisms very different from us: bacteria and archaea. In these relatively simple cells, CRISPR-Cas is an analogue of adaptive immunity. CRISPR itself is an abbreviation describing a section of the bacterial genome where information about those viruses that the ancestors of this cell encountered is recorded. This data is stored in the form of a library of short pieces of viral DNA, which the bacterium inherits and can replenish independently.
If a bacterium encounters a virus, information about which is recorded in the CRISPR library, Cas proteins can recognize and destroy the "uninvited guest". To do this, it is necessary to present the data from the library in the form of an RNA molecule. The Cas-RNA complex scans DNA and looks for matches, and if there is a match, it cuts.
What does human genetic diseases have to do with it? The key here is the ability of Cas proteins to cut DNA in a well–defined area. The idea is as follows: instead of reading RNA from the CRISPR library, scientists simply take the necessary short RNA molecule (it is called a guide, or guide) corresponding to a specific place in the genome. In combination with the Cas protein (of all the natural diversity of CRISPR-Cas systems, the Cas9 protein from streptococcus is most often used to work with animal cells), the guide RNA is injected into cells. There Cas9 finds the desired area, for example, containing a mutation, and makes a cut.
However, this is not all. To correct a harmful mutation, in addition to the Cas9 protein complex with RNA, another "patch" containing the desired DNA sequence must be added to the cell. Using it, cell repair systems will "repair" the cut DNA and instead of mutation, another "normal" sequence will appear in this place.
It is necessary not only and not so much for treatment.
Genome editing technology opens up new horizons for its researchers. The ability to vary the DNA sequence of living organisms existed before, but compared to the genetic engineering methods that existed before the "CRISPR-Cas era" (which, by the way, began only six years ago), the proposed mechanism is quite simple and effective. It helps to quickly create model systems for a very different class of tasks, both in the field of fundamental genetics and in applied medicine, and has already become a kind of must have in many biological laboratories.
Why are the results of He doubted?
Doubts arose because of the form in which the new result was announced. Firstly, He did not publish his work in a scientific journal, violating the usual procedure for announcing the results of experiments. An article in a journal is usually read and evaluated by several reviewers and an editor before publication.
Instead, He recorded a video on YouTube, in which he announced not only the success, but also that the girls who were born could not be seen, and the data about their family was classified. The Southern University, where the scientist is formally registered, added fuel to the fire – they said that he had been on unpaid leave for six months and they did not know anything about this work.
Secondly, there are doubts of a more general kind: why, asks the famous scientific journalist Leonid Schneider, was HIV chosen for such an epoch-making experiment, and not some kind of congenital fatal genetic disease?
I am in solidarity with Schneider and Paul Kalinichenko is a professor at the Kutafin Moscow State Law University (MSLA), who studies the world practices of legislative regulation of genetic experiments. "This is a very strange example. HIV is not a genetic disease, that is, when editing the genome, there is no treatment, but only a reduction in the risk of infection. But HIV is a very well–known disease. 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 with HIV is possible, it is a pandemic, a kind of tribune. That's why I doubted the authenticity of [He's statements]," Kalinichenko says.
Is the birth of genetically modified people even possible?
Yes, quite – and most experts agree with the practical possibility of such work. Moreover, He is in one of the best places to conduct such research.
"It is difficult to verify what has been said, but assessing a hypothetical possibility, we can rely on the history of previous years. And we know that it was Chinese scientists who edited the first human embryo genome – this experiment was conducted back in 2015 (though not too successfully). There we were talking about non-viable zygotes, that is, the embryo was not planted by the mother. A year later, our famous compatriot Shukhrat Mitalipov, who now works in University of Health and Sciences of Oregon, developed and consolidated this experience," says Pavel Volchkov, head of the Laboratory of Genomic Engineering of the Moscow Institute of Physics and Technology (MIPT).
Mitalipov, according to all the canons, published his article in Nature. It proves the possibility of editing the human genome at the embryo stage in order to avoid the manifestation of a genetic disease – hypertrophic cardiomyopathy, for which today there is only symptomatic treatment. The gene–editing drug was injected into the zygote - embryo at the stage of its unicellular development. Then the zygote was allowed to develop to a blastocyst – the first multicellular stage. By analyzing the genome of the cells, it was shown that the editing took place. At this point, the experiment was interrupted.
"As you can see, all the fundamental work was done, it remained only to plant this blastocyst back to the mother – that is, to perform a completely routine operation, common with IVF, which women use, for example, with obstruction of the fallopian tubes. Why was the experiment always interrupted earlier? In order not to conduct an illegal experiment on a person. The fact is that experiments on embryos are legal, since in different countries he is not considered a person until a certain age. It was up to this age stipulated in the law that the multicellular stage was raised," explains Volchkov.
How difficult is it?
Apparently, it is not very difficult to bring out genetically modified people - of course, in the conditions of a modern laboratory engaged in genome editing and, preferably, working with a large reproductive clinic.
"The technology of microinjection into a fertilized zygote, with the help of which genome editing is carried out, is not difficult,– says Pavel Volchkov. – And He worked in the laboratory where IVF is done. In such a laboratory, there is always a large number of fertilized eggs at hand from parents who are trying to give birth – usually more eggs are taken for IVF than necessary, in case of failures, and they remain in the clinic. This means that there is always an opportunity to stab genetic editing tools into zygotes, let them develop to a certain stage and evaluate the effectiveness of this procedure."
"The technique consists of several procedures. Embryological procedures – working with an embryo, with a zygote, with a micromanipulator, injection – can vary from laboratory to laboratory. He, at least, according to him in the video, carried them out in the same way as we do in our work," says Denis Rebrikov, a geneticist, vice–rector of the Pirogov Russian National Research Medical University (RNIMU), head of the genome editing laboratory at the Kulakov Scientific Center. Previously, a scientific group under his leadership conducted almost the same experiment with human embryos, with the only difference that the edited eggs were not planted to the mother.
According to Rebrikov, we are talking about a standard procedure for the treatment of male infertility according to the ICSI protocol (ICSI, Intracellular Sperm Injection Protocol), used when the sperm are too immobile for conception: "Simultaneously with the sperm, we micromanipulate a mixture for gene editing into the egg, thereby obtaining a zygote," says the scientist.
"Standard purchased Cas9-type enzymes are usually used for editing. There are quite a lot of variants of enzymes for today, so it is impossible to say which enzyme He used. But the rest of the components of the reaction mixture: guide enzyme guide RNA, oligonucleotides and a special "DNA patch" (a fragment of DNA acting as a template in the sewing process) - as a rule, each laboratory is made independently," continues to explain Rebrikov.
The mutation that was introduced into the embryos is also not completely new. Moreover, it is not artificial either – about one percent of the inhabitants of Europe are innately resistant to HIV, that is, they carry two alleles of this mutant gene, and 10 percent carry one allele.
"This modification corresponds to a variant of the gene present in the population, which is an allele that arose during evolution, a variant of the gene without 32 letters. And there is some ethical relief in this situation, because we are not saying that we have created a new allele, a new variant of a gene that does not occur in humans. Thousands of people were born in a completely natural way and live with just such a variant of the gene," emphasizes Rebrikov.
How dangerous is it?
The technique has already been tested, but the transition from the laboratory to clinical practice is a completely different matter, and it takes pharmaceutical companies years, if not decades. From the purely technical side of the matter, in order to ensure security, you need to be sure of two things: the method effectively edits the target DNA site, and this happens on a statistically significant sample with a small percentage of failures (editing of the target locus), and at the same time other parts of the genome are not edited (non-editing of a non-specific locus).
"Given the scale of the center He worked with, this technique was most likely practiced for three to four years. They gathered information on embryos and, based on their statistical data, allowed themselves to set up this kind of experiment," Volchkov suggests.
By itself, HIV genetic therapy is also not absolute news. Songamo is testing this method to treat the virus, but only on somatic, "normal" cells, not embryonic stem cells. It came to clinical trials, which means that a lot of data has been accumulated on the problem. These are both company data and open data in scientific publications.
"This gene and the targeting system are well studied, the Chinese did not do it blindly, they only transferred this technology to the editing of the embryo, not somatic cells," Volchkov notes.
However, he is not sure of the absolute correctness of the procedure performed.
"What would I like to see to make sure that the work is correct? First of all, these are preliminary experiments on cell lines (embryonic table cells). A statistically significant number of experiments on embryos with interrupted development – for example, 25-50 cases, where it is clearly shown that a targeted gene is being edited and there is no or almost no non-specific targeting of other alleles that could make a negative contribution to the condition of the future person. Only after that it would be possible to move on to the next phase," says Volchkov.
But, according to him, there is a question about the legal regulation of such experiments. "Only the regulator, in this case the Chinese equivalent of the FDA, can establish the criterion of this "almost absent". While there is no criterion, it is difficult to reason what is permissible and what is unacceptable," the scientist argues.
"But let's imagine that this stage has been passed. Next, I would like to see tests on an animal model. The most closely related human model is the great ape. If genetic editing had been demonstrated first on them, and not on human twins, it would have been more correct," Volchkov continues. – Moreover, work in this area is already underway: This year, Chinese scientists published an article in Nautre that they cloned a macaque (non–human monkey), and another one that they edited its genome."
But He and his group, apparently, do not want to waste time on experiments on monkeys. "The fact that they missed this important stage and moved on to human experiments does not speak in their favor," Volchkov concludes.
At the same time, human embryogenesis is a highly self–regulating system, and if something goes wrong in it, then embryonic development is terminated (miscarriage occurs at one stage or another of pregnancy). However, this mechanism, unfortunately, does not always work, the scientist notes. But if you rely on the high probability of its work, it means that the very fact of the birth of girls confirms the integrity of their genome.
Konstantin Severinov, a professor at Skoltech and Rutgers University, notes that conclusions about safety, strictly speaking, can be made only by bringing the experiment to its logical end: after genetic manipulation with an egg, a child should be born, grow up, produce their own children, live a more or less normal life. "So it was with Dolly the sheep. The success of the experiment with her, in particular, was that she gave birth to another sheep. But people have a life span comparable to that of researchers. In this sense, it is very difficult to set up an experiment so that it corresponds to the level of evidence that you want to have before using the procedure," the scientist said.
What exactly has been done
On November 28, Jiankui He made a presentation at the GeneEdit Summit in Hong Kong, where he apologized for the early "information leak" and told about the technical details of his work (the transcript of the report and presentation slides were posted by the conference visitors on Twitter).
So, the aim of the work was to introduce into viable human embryos a natural CCR5-delta32 mutation, that is, a deletion of 32 nucleotides, which disrupts the gene and protects its carriers from HIV infection.
According to He, before editing human embryos for transplantation, they carefully selected the guide RNA and tested it on non-viable embryos and embryonic cell lines. In addition, using the selected "seed", the He staff raised a macaque with the desired mutation in the genome.
The most important part of He's report was the DNA analysis of the edited twins. After birth, DNA was isolated from the umbilical cord blood of the girls and their genomes were completely sequenced. In addition, DNA was isolated from several other tissues. As a result of sequencing, no non-targeted Cas9 activity was detected.
Has the stated goal been achieved? The sequences of the CCR5 protein in both girls really do not coincide with the "wild type" protein. However, apparently, they do not have the necessary mutation (delta-32) either. The slide shows that one of the girls had a deletion of 15 nucleotides in one copy of the gene, that is, five amino acids, and the other copy remained intact. Thus, the CCR5 protein has lost a small piece, but can still be functional.
Her sister had both copies of the gene affected – in one of them a small deletion of four nucleotides, and in the other a single nucleotide insert. The protein in both cases will be shortened, but it is unclear how this will affect resistance to the virus.
The CCR5 sequences of the girls indicate that the editing achieved its goal only partially – the guide RNA worked, the Cas9 protein made the incision, but the cellular repair systems, instead of using the necessary "patch", healed the incision indiscriminately. In addition, both girls, apparently, turned out to be "mosaics", that is, some of their cells remained unedited.
Scientists have encountered these problems in all published articles on editing human embryos, and the result of the first experiment on humans confirms: no matter how interesting it is to test the breakthrough technology "in battle", it is still not sufficiently developed to accomplish the revolution.
Comparison of the original and edited genetic code of Lulu and Nana (Sean Ryder).
Is it ethical?
Experts believe that it is not. And for several reasons at once.
According to Oksana Moroz, Associate Professor of the Department of Cultural Studies and Social Communication of the Russian Academy of National Economy and Public Administration (RANEPA), there are two approaches justifying the morality of certain actions – deontology and consequentialism.
"Deontology suggests making a decision about morality based on the rules adopted in society. If we follow the rules, it's moral, if not– then no. The whole story is that experimenting with embryos for up to seven days is ethical, and after that it is unethical, because after that the embryo is attached to the uterus, this is a purely deontological approach. Consequentialism evaluates the morality of an action through its result. If the consequences are good, then it's good. And it is to this approach that the Chinese scientist appeals. It doesn't matter how he says that I proceeded from moral premises that are not accepted by everyone. It is important that the consequences of my actions will be wonderful," Oksana Moroz explains the discussion frame.
But the question of global consequences in this case goes beyond concern about the health of specific people. We are talking about the integrity of the genetic base of the human population, the scientist notes.
"When we edit the genome and sort of construct a new person, we can't be sure that we edited exactly what needed repair and didn't damage something else important in the long run. At first glance, there seems to be no harm from such manipulations, but since there is no longitudinal data, we cannot judge how much our intervention in the genome helps only to heal and does not cause any delayed harm. After all, in the future, these genetically modified people will fall into the volume of the genetic material of mankind, they themselves will become parents. And it is possible that our invasion of the common genetic space may have a delayed effect," Moroz notes.
In addition, there is also the issue of informed parental consent. He himself has already stated that the people he subjected to this experiment (the parents of the twins) knew what they were doing. But there are doubts about this, too. "It is recorded on paper that he is conducting an experiment on HIV therapy, and only verbally he told his parents exactly what he plans to do. And the big question is how far people who are far from genetics could understand the essence of his actions," the scientist says.
The potential problem of elitism is also of concern.
"We can proceed from the fact that we will cure everyone – even at the stage of embryonic development, when future people still do not feel anything and do not suffer. But if our technology is implemented and released to the market, then, most likely, it will not be available to everyone. And then some X-men will appear in society, a new type of inequality will arise – not social and not physiological, inherited from birth, but inequality based on artificially made physiology, – says Oksana Moroz. – And it is not very clear how we will cope with this, because we are already struggling to cope with the fact that there is gender diversity and a third gender. How ready is society for the appearance of characters with this type of features?"
Finally, this is an experiment on humans.
"The scientist wants praise, but does not think that as a result of his actions, living people will be born who will think, and feel, and understand that they were born as a result of an experiment. This is a story about Frankenstein's monsters, which are created, and then tormented by the fact that they are the fruit of human pride. Religious and moral factors are intertwined here, which call into question the legitimacy of such actions. It is no accident that Chinese institutions distanced themselves from this work," the expert notes.
However, Denis Rebrikov from RNIMU says that it is too early to think about the problem of the appearance of "superhumans" as a result of genetic editing. So far, in all cases, the genetic modification of embryos has been limited only to those genetic variants that already exist in the population. "We know that there are people with a deletion in the CCR5 gene, there are quite a lot of them in Northern Europe, that this is not a mutation that entails serious consequences. No one is going to insert any letters into the human genome yet, which do not happen in it," he explained.
Rebrikov emphasizes that in such experiments, the genomes of both parents and the genome of the resulting embryo should be sequenced before its transfer to the expectant mother. In this case, you can be sure that only the planned DNA sections have been changed and no other genetic changes have occurred.
Is it forbidden?
Here we turn to the deontological approach of ensuring morality.
"We are talking about an area with poorly developed legal regulation. It accumulates around prohibitions and restrictions, but procedural norms that allow positive solutions to certain issues are minimal," says Paul Kalinichenko from the Moscow State University.
That is why such experiments are more possible in the countries of Southeast Asia, where regulation in this area is weaker than in Europe or the United States. In Europe, ethical barriers are deeply embedded in legal practice, so the case in question falls into a broader category – "experiments on people". They are categorically prohibited, and this is historically predetermined. "In Europe, this refers to eugenic research, and in EU eugenics is banned altogether, even positive eugenics. Therefore, such work is impossible there," the expert concludes.
Great Britain stands apart in this row. There, work on editing the genome of embryos was allowed in 2016 after China – of course, under the supervision of ethical and research commissions.
"The situation is milder in the USA, but it does not mean that Americans can afford such experiments right now. There, the law varies from state to state, and still the legal regulation has not fully developed. In addition, the American approach is, let's say, practice–oriented. A certain gap in the legislation is laid specifically so as not to hinder the work of the industry where there is commercial potential. Full-fledged regulation is postponed for later: if something goes wrong, they will do it. In the meantime, everything is harmless, let it develop," Kalinichenko explains.
This approach can also be seen in the regulations governing work with embryonic stem cells: restrictions apply only to research that receives federal funding.
"China is a non–legal state, so it is impossible to fully understand the meaning and goals of the state's policy. It is utilitarian in its goals and objectives, that is, it obeys the tasks of the CPC. But what is important is that the Chinese authorities support scientific research, allocate huge amounts of money to outbid scientists, create conditions for their activities. Apparently, the result is important for them and this may explain a certain liberality of legislation. That is, the concessions that the Chinese make serve the purposes of science, not business. If it comes to clinical practice, the liberalization of laws can quickly be replaced by strict regulation," the scientist notes.
In Russia, regulation in this area has been lowered to the local level. Much here is determined on the basis of existing practices and norms of specific institutions, is not subject to any rigid circulars. The expert does not undertake to assess whether this is good or bad, but calls for the improvement of Russian legal reality based on an objective analysis of needs, including scientific work.
Will such operations become commonplace soon?
Apparently, it is not so important whether the genetically edited babies were born right now. If this has not happened yet, it can happen at any time, and it will happen for sure.
"It's only a matter of time before this is done. Whether it's He or someone else, it doesn't matter. There is such an expression: it's not a question of if, but when, – "the question is not whether it will happen, the question is when it will happen." I think the person who does it first and succeeds will have a very difficult time at first. But then he will be carried on his hands," Konstantin Severinov concludes.
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