17 March 2016

A genetically modified person

Why do scientists want to change the genome of human embryos

Natalia Nifantova, "The Attic"

In February 2016, experiments on editing the genome of human embryos were allowed in the UK for the first time. This case was already the second time when genetics is taken into the holy of holies – the "original" DNA, on the basis of which all other human cells are created. "The Attic" found out why such experiments are needed and why they are dangerous.

The birth of a new human life is a real miracle, even from the point of view of science. In a single cell, the halves of the genome of the father and mother first merge, and then this set of 46 chromosomes creates all kinds of cells of the future organism: from auxiliary cells of the placenta and umbilical cord to osteoblasts, from which bones are built, and photosensitive cells of the retina of the eye. At the same time, each type of cell "knows" the time and place of its appearance, otherwise a cell soup would have turned out instead of a new person. The amazing accuracy with which cells determine the "schedule" of development is achieved due to the fact that DNA and its assistants – RNA and proteins – work like a well-played orchestra, smoothly and accurately regulating the activity of genes.

It is not surprising that since scientists learned to decipher DNA and RNA sequences in the 1970s, the Holy Grail of molecular genetics has become the opportunity to find out exactly what happens to DNA during embryonic development, which genes are responsible for making a whole person out of a lonely little cell. But until 2012, there was no suitable tool for such research.

"Some aspects have been studied, but mostly it is a dark forest," says Corresponding member of the Russian Academy of Sciences, Doctor of Biological Sciences, Head of the Laboratory of Molecular Biology of Stem Cells of the Institute of Cytology of the Russian Academy of Sciences Alexey Tomilin.

There are two main ways to find out what function a gene performs – turn it off (this is called a gene knockout or knockdown, by analogy with a boxing punch, after which the opponent cannot continue the fight) or replace it with another (transgenesis) and see what will change in the life of the cell and the whole organism after that.

Such manipulations with the genome are traditionally carried out on embryonic stem cells (ESCs) of mice, which are then injected back into the embryos, and those, in turn, are planted in the uterus of mice for implantation. As a result, chimeras are born, animals, some of whose cells carry modified "donor" DNA, and others – the DNA of a surrogate mother. The effect of the modification introduced into the genome is studied on their descendants, some of whom will be carriers of only the modified genome. "The use of such an approach to study early human development is obviously impossible," Tomilin explains. "The only way to carry out gene manipulations with a human embryo and assess their impact on its development is a short period of six days between fertilization and implantation."

Until recently, scientists also faced a purely technical problem. To edit the genome, you need to make the enzymes-nucleases that break down the DNA chain, contact it strictly in the right place. The "guidance" methods that were used earlier coped with their task in about 20% of cases.

This is quite enough to create genetically modified plants, conduct experiments on mouse embryos or cells of "adult" human tissues. In all these cases, you can take a lot of experimental cells at once, and then select for further use only those in which the editing was successful. But human embryos are too valuable an object for research. They can only get into the scientist's laboratory as a gift from couples who have undergone IVF (several eggs are fertilized at once, but one or two are implanted into the mother, the rest remain in storage in freezing or are destroyed). Given the inaccuracy of genome modification technologies, such a number of eggs is categorically insufficient.

"The situation changed radically after the discovery of the CRISPR/Cas9 gene editing technology," Tomilin says. The CRISPR/Cas9 system, first tested in 2012, by 2015 showed an efficiency of 90% on mouse embryos and 94% on immature human T-lymphocytes and hematopoietic stem cells. It would seem that it's time to go on a campaign for the Grail.

Ethics stopped

In April 2015, for the first time in the world, experiments on editing the embryonic genome were conducted by Chinese scientists from Sun Yat-sen University under the leadership of Junjiu Huang. They took 86 fertilized human eggs and, using CRISPR/Cas9, tried to correct a mutant gene in them that causes beta-thalassemia, a severe hereditary blood disease. The result was unexpected. CRISPR/Cas9 correctly changed the genome in only 28 embryos, and during further division, only four of them retained the new gene. However, this did not stop the Chinese researchers. Junju Huang is going to continue experimenting with human embryos, primarily to find ways to increase the effectiveness of CRISPR/Cas9.

"Huang's research has shown that it is too early to talk about editing the human genome at the preimplantation stage," explains Alexey Tomilin. – Too low efficiency and too high risk of side changes in the genome (the so-called off-target effect). When both problems are solved, then it will be possible to talk about the genetic correction of the human germ line. It is difficult to say why CRISPR/Cas9 often misses the target in the embryonic genome. Work is underway to improve the accuracy and efficiency of editing using CRISPR/Cas9. There is no doubt that there will be progress."

The article by Chinese researchers unexpectedly provoked a loud response from their European and American colleagues, and scientists were not at all concerned about the low accuracy of editing, but the ethical side of the issue. Already in April 2015, a response article appeared in the journal Science signed by 18 genomics and stem cell specialists, among whom were researchers who were directly involved in the development and improvement of the CRISPR/Cas9 method – Jennifer Dudna and Martin Ginek. They urged their colleagues to be cautious about the prospect of editing the embryonic genome, insisting that people need time to comprehend the possible consequences of such an intervention, otherwise eugenics is not far away – breeding a "breed" of people with given characteristics. The concern of the authors of the article in October 2015 was supported by the International Committee on Bioethics at UNESCO, calling for a temporary moratorium on such work with human cells.

What are scientists so afraid of? Ethical issues are not caused by suffering or destruction of embryos during genetic experiments. At the stage of one to six days after fertilization, the embryo is a lump of only a few dozen cells. The concern is precisely the non-destruction of modified embryos. Changes made to the genes of germ cells, fertilized egg cells and embryo cells in the early stages of development are inherited by all descendants of the modified organism. This is called a change in the germ line.

The first step

Despite the ambiguous results of Junju Huang's group and the ethical dilemma of genetic editing of embryos as such, on February 1, 2016 it became known that the British Human Fertilization and Embryology Authority (HFEA – Human Fertilization and Embryology Authority) granted permission to edit the embryonic genome to Dr. Cathy Niakan from the Francis Crick Institute.

Niakan has been studying for almost 10 years how stem cells are determined with their future specialization in human and mouse embryos. Recently, her research group has been trying to find out the answer to this question by deciphering sequences of RNA–intermediary molecules that transmit information from DNA to ribosomes, cellular machines that synthesize proteins. Scientists have managed to identify several genes that work only in human cells and determine differences in early human development from the same mice, for example, the KLF17 gene. To understand what functions these genes perform, experiments requiring DNA editing are needed. In this sense, the goals set by Niakan and her colleagues are much closer to the search for the genetic Grail, that is, to answers to fundamental scientific questions, than the goals of Chinese scientists.

Another task of British biologists is to understand which genes are responsible for the successful development of the embryo as a whole, and especially for the correct formation of the placenta. This knowledge can change a lot in the diagnosis and treatment of infertility. Statistics say that 15-20% of all pregnancies end in miscarriage at the earliest stages, while women do not even know that they were pregnant. On the other hand, during the IVF procedure, only 25% of embryos are successfully implanted into the uterus of the expectant mother. Most often, this is due to the genetic problems of the embryo itself, which at the right moment cannot attach to the wall of the uterus or later form a full-fledged placenta for its development. Niakan also has its own "suspect" here – the Oct4 gene, the lack of activity of which in mice is associated with slowing down the production of stem cells.

A human embryo at different stages of development.
The cells in which the genes marked on the left are active are highlighted in the appropriate color.
Photo: Kathy Niakan group, Francis Crick Institute

The third goal of Niakan is to understand how the development of embryonic stem cells (ESCs) in vivo differs from their growth and specialization in vitro. Replacement therapy with embryonic stem cells is both a very promising and very dangerous method. Promising – because ESCs do not cause an immune response that leads to rejection of donor tissues during a routine transplant. In addition, cells of any organ can be grown from ESCs. In the future, they can be used to treat Alzheimer's disease, coronary heart disease, thyroid insufficiency, cerebral palsy and much more.

This method is dangerous because outside the embryo, stem cells often behave unpredictably. For example, in experimental animals, they cause the formation of tumors. To turn such consequences, it is necessary to find out which genes in the ESCs in vitro work differently than in the embryo, and what conditions affect this. Again, Niakan and her team already have candidate genes, for example ARGFX.

British biologists will have to deal with all these issues in a short time – the HFEA permit is valid for only three years. And this is not the only restriction imposed on the Niakan project. During the experiments, the embryos can develop for only 14 days, after which they must be destroyed.

What's next?

The sequential activation and termination of certain genes in the process of embryonic development is not just written in the DNA, it is influenced by environmental factors – the hormones of the mother, substances entering her body from the outside. At the same time, it is known that in mammals, the conditions in which the embryo developed can determine the future fate of the born creature – to program certain diseases or a tendency to them, for example hypertension or metabolic syndrome.

For humans, many of these factors are not even described, because no one will conduct experiments on pregnant women. DNA editing technologies are still too imperfect to breed genetically modified people, but with their help it is already possible to find out where congenital diseases come from and how to prevent them. According to Alexey Tomilin, the "green light" for Katie Niakan's project is the first, but not the last "relaxation". In those countries where experiments with preimplantation human embryos are not directly prohibited (this is the case, for example, in Germany), new research projects will surely appear soon, seeking to look into the holy of holies.

Portal "Eternal youth" http://vechnayamolodost.ru  17.03.2016

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