02 December 2016

Scissors for the genome

CRISPR/CAS9: What does the transition from reading the genome to editing it mean for humanity?

Artem Elmuratov, Co-founder of Genotek
Dmitry Korostin, Director of Science at Genotek
Forbes, 01.12.2016

How scientists modify genes, what ethical questions this may raise and whether "custom-made children" will be created with the help of gene technologies.

CRISPR/Cas9 technology, which allows making changes to the genome of higher organisms (including humans), has become one of the most discussed topics in recent years – not only by molecular biologists, but also by biotech investors, doctors, sociologists. The thing is that CRISPR/Cas9 potentially has prospects of application to combat many serious diseases, including cancer, hereditary diseases, HIV. If earlier genetic technologies were used primarily for diagnostics, now for the first time we have reached a new milestone – we have a DNA editing tool that may receive more and more implementations in clinical practice and treatment programs. Although earlier attempts at genome editing have already been made (for example, for patients with leukemia), it is CRISPR/Cas9, as a more versatile tool, that claims to create tools for increasingly active implementations.   The start has been given: China is already taking the first steps in clinical trials of technologies based on CRISPR/Cas9.  The growing possibilities of gene therapy pose more and more questions related to ethics. Why wait?

CRISPR/Cas9 – "scissors" instead of "knife" for DNA

Methods of gene editing in the genomes of living beings have been studied since the beginning of the XX century – since the discovery of the mechanism of induced mutagenesis (that is, caused by the influence of some external agents - for example, radioactive radiation or chemicals). And if the mechanisms of sufficiently precise gene modification for bacteria were developed in the middle of the XX century, then for more complex organisms, in particular, humans, approaches appeared only at the end of the last century. For example, a whole family of viruses called retroviruses (which includes HIV, which causes AIDS in humans), has received a mechanism by nature, according to which the functioning of the virus requires embedding its genome into the genome of the host organism. By introducing modifications into the retrovirus genome, that is, inserting modified human genes, it is possible to achieve the introduction of such quasi–human elements into the host genome - that's the ready-made genomic editing mechanism. Its significant disadvantage is the lack of specificity of embedding, that is, the viral genome can get into any part of the host genome, or it may not get at all. For scientific research, this is not a big deal – you can always repeat the experiment. But for the purposes of treating specific patients, the "repeat" approach usually does not work.

Other methods of genome modification are related to ZFN and TALEN technologies, which have been actively discussed since the 2000s. The idea of these approaches is also based on the natural properties of certain proteins called nucleases. These active proteins (enzymes) are able to carry out a specific, non-random excision of a section of the original genome and embedding a piece of corrected DNA brought with them into the incision site. This method allows for targeted, much more accurate than just retroviral, modification of "broken" genes. The difference between ZFN and TALEN is the use of different types of enzymes, but the result of their work is approximately the same.

In 2015, ZFN technology was successfully applied to HIV therapy: the site responsible for HIV susceptibility was edited in the donor's stem cells, then they were transplanted to the patient. It is worth noting that approximately every thousandth European has such a genotype that guarantees the impossibility of introducing a virion (an active viral particle that carries out infection) into the cells of the host organism, that is, the impossibility of infection.

If you cut the DNA sequence in two places, then you can use the natural mechanisms of repair of its chain - do not let it connect to another DNA or just "stitch" the two ends, and add the necessary fragment, introduced in advance, to the place of rupture.  The site of the incision helps to localize special proteins that are tied to certain gene sequences and binding sites of the genome. It turned out that such proteins have areas with zinc ion, which make them even more specific to the DNA fragment – correlates them with only three nucleotides. If you attach these "zinc fingers" of genetically engineered proteins as identifiers to the "scissors" (their role is performed by a special kind of nuclease, an enzyme for reactions between nucleic acids), you can accurately identify the gene sequences on both sides of the desired "cut". This is how ZFN (Zinc finger nuclease) technology works. TALEN (Transcription Activator-Like Effector Nuclease) technology is based on similar principles, only TAL proteins are used to create a DNA–binding site of a protein that "knows" the desired gene sequence.

But ZFN and TALEN turned out to be far from mass use in medicine. Scientists tried to teach them to recognize a specific, ideally any given DNA sequence for "biting". Sometimes it worked, but for each sequence it was necessary to create its own separate protein, and this is a painstaking and long work.

Another thing is genome editing using the CRISPR/Cas9 system. It uses a single protein (CAS protein complexes), and a "guide" for finding a DNA site for the necessary modifications can be done very quickly and cheaply.   Due to certain sections of the CRISPR genome that store the genetic sequences of viruses, the CAS protein cuts the corresponding sequences. Many, many well-equipped laboratories can work with this. That is why CRISPR/Cas9 technology, originally discovered as a protective mechanism of bacteria against viruses in the late 1980s, caused such a stir.

It is not yet clear which genomic editing technologies will be most actively used in medicine in 10 years. Perhaps it will be CRISPR/Cas9 or current analogues, or maybe a new technology will be discovered that will arise as unexpectedly and vividly as CRISPR/Cas9.

In the meantime, there is a patent war going on between two scientific groups that in 2012 simultaneously found a way to use CRISPR/Cas9 for point editing of the genomes of complex organisms. A group at the University of California at Berkeley and a group from MIT and the Broad Institute (MIT and Harvard Institute), who filed patent applications at different times in 2013, are spending tens of millions of dollars on lawyers and are unlikely to stop – billions of dollars that technology can bring are at stake. According to forecasts, the patent office will make a decision in 2017.

Venture investors are already actively investing in companies developing CRISPR/Cas9. In 2016 alone, three such companies went public. The capitalization of the first, Editas Medicine, is $542 million, and Bill Gates is among its investors before entering NASDAQ. The second, Intellia Therapeutics, reached a capitalization of $ 600 million, and among its first investors was the Swiss pharmaceutical giant Novartis (No. 47 in the Forbes global list).The third, CRISPR Therapeutics, has so far passed the mark in capitalization of only $ 107 million, the pharmaceutical company Bayer (No. 97 in the Forbes global list) participates in its financing.

Application and ethical issues

Among the potential applications of the new technology are the treatment of hereditary diseases (hemophilia, beta–thalassemia, muscular dystrophy), therapy of oncology and viral infections, including HIV.

But there are also more exotic potential uses. For example, the fight against multifactorial diseases (diabetes, schizophrenia, etc.) or editing embryos during artificial insemination to select a certain appearance in children. This is where a lot of ethical issues arise, which have begun to be discussed, but have not yet received a consensus solution from the world community. When is it possible and when is it impossible to use genome editing? So far, in the absence of a common position among mankind, each of the countries decides this in its own way. 

For example, two studies by Chinese scientists have already shocked the world. In 2015, a group of Chinese scientists applied genomic editing on 86 human embryos in order to change mutations that cause severe hereditary pathology beta-thalassemia. This is a severe hereditary pathology, which is associated with a violation of hemoglobin synthesis and destruction of red blood cells, the average life expectancy of mutation carriers is 17 years. Despite the serious scientific significance of the study by Chinese scientists, the two main scientific journals Nature (UK) and Science (USA) refused to publish the results, in particular due to ethical issues.  Also, this study showed the imperfection of CRISPR/Cas9 technology, at least at the moment. Out of 86 embryos, only 28 managed to accurately change the desired DNA site. The percentage of error turned out to be greater than the researchers expected based on experiments on animal embryos. Which part of the DNA needs to be edited is determined using a synthetic RNA sequence (the so-called "guide").  It is complementary to the desired DNA site. But it may turn out that there is also a similar sequence of nucleotides in another part of the genome.

The study has caused a lot of discussion. Should Western countries be very careful about ethical issues when applying new genomic editing technologies, or will this only lead to lagging behind China? Apparently, while the West is considering the possibility of being more tolerant of genetic modifications – less than six months after the scandalous publication of Chinese researchers in the UK, the first experiments on genomic editing of human embryos were officially allowed, in London new groups of scientists are already working on the design of new experiments.

And in mid-November, a group of Chinese scientists announced the use of gene modification of cells using CRISPR/Cas9 to treat a patient with aggressive lung cancer. In the journal Nature, this article was announced with the subtitle: "A move by Chinese scientists could ignite a struggle between China and the United States in the field of biomedicine."  The world is waiting for the results of the second study by Chinese scientists.

However, in the future, there will be even more ethical issues related to new technologies in the field of genetics and reproduction.  Bioethics is becoming an increasingly important discipline.

For example, some Europeans have mutations in the CCR5 gene – its carriers are practically immune to HIV. As part of genetic testing, these mutations can be investigated. But is it ethical, is it correct to tell a person about the presence of such a mutation? We, as a company that deals with genetic tests, decided that no.

Other problems are related to the fact that the possibilities of genetic editing change the very concept of family.  With the advent of artificial insemination and surrogacy, in principle, the understanding of the institution of the family has become more complicated. Now some children may have two mothers besides their father: a surrogate and a "legal" one.  And if an egg and a sperm of a couple are used for conception, which are then transferred to a surrogate mother, the child also has two mothers – a genetic and a surrogate. Theoretically, there are situations when the legal, genetic and surrogate mother are three different people.

The legal nuances that arise in such cases are already being evaluated. For example, "Baby M Case": Elizabeth Stern had multiple sclerosis, which carries many risks during pregnancy, so the Stern family turned to one of the medical centers in New York for surrogacy.  The father's genetic material was used. An agreement was signed between the parties that the legal parents would be the Sterns. But soon after the birth, Mary Beth Whitehead's surrogate mother demanded to return the child to her under the threat of suicide. The police and the court got involved in the case. As a result, the court recognized the Sterns as the official parents of the child, but gave the surrogate mother the opportunity to visit the child. Interestingly, the main motivation of the court was the pursuit of the "best interests of the child".  In another similar story, which also happened in New York, the court judged differently:  two parents are better than three, the court decided, denying the rights to visit the surrogate mother's child. There is a detail: in the second case, the couple used their own egg and sperm, which were transferred to a surrogate mother.

"Offshore" for genomic editing

Recently, the world was shocked by the birth of a child from three genetic parents: in April 2016, a child was born, whose conception took place using the mitochondrial DNA of a third person. Such a procedure was necessary, since the child's mother has pathogenic mutations in the mitochondria (organelles inside human cells responsible for providing cells with energy, have their own small genome, are transmitted to the child from the mother), which could lead to the appearance of Ley syndrome in the child, a hereditary disease associated with CNS damage and encephalopathy. The mother's first two children died from Ley syndrome. American doctor John Chang, from a clinic in New York, arrived in Mexico with his parents from Jordan.  In both Jordan and the United States, such modifications of genetic material were prohibited.

It turns out that "biomedical offshore companies" are appearing in the modern world. People travel to countries with loyal legislation to carry out procedures that are ambiguous from the point of view of ethics and permissibility by the legislation of a particular country. Gene therapy is already becoming the focus of such "controversial" cases. For example, American Liz Parrish claims that she underwent a procedure in Colombia to edit special sections of DNA-telomeres using a virus. Telomere length correlates with aging. Parrish became the first person to decide on genetic therapy to combat aging, before her experiments were conducted only on animals. The scientific community reacted ambiguously to Parrish's unauthorized clinical trials, many criticized her actions.

In general, at the moment, the world community is very cautious about genome editing when it is not directly related to the treatment of serious diseases that cannot be cured in any other way. The fact is that the technologies are still imperfect and not as specific as possible.  So, in the already mentioned experiment of Chinese scientists on embryos, in the DNA of many embryos, not only the areas that scientists planned to change, but also others, random ones, changed. Or, for example, when in France it was decided to use gene therapy to treat congenital X-linked immunodeficiency, during a clinical trial, leukemia unexpectedly developed as a side effect in the patient. 

Child to order

In general, the medical and scientific community is now more loyal to gene therapy, which will affect only the genetic material of the person himself. Gene modifications that would be passed on to children are still insufficiently studied and remain in the "gray zone". But to a certain extent, the choice of certain traits of a child with the help of genetic technologies is already available now.

During artificial insemination, preimplantation genetic diagnosis or preimplantation genetic screening can be performed. With IVF (artificial insemination, outside the mother's body, followed by the transfer of a 2-5-day embryo into the uterine cavity), several eggs are fertilized today. You can examine the genome of each of them and choose the most "suitable" embryos. This procedure is already quite actively used for the prevention of severe hereditary pathologies in families with corresponding risks. However, this technology can obviously be used to select traits that are not related to health – for example, eye color or hair. This is certainly a somewhat frightening situation, it makes you think about ways to use new genetic technologies for eugenics, for other manipulations described by science fiction writers in dystopias.  Different countries are already developing a position on the influence of parents on the genetic data of their children.

In China, for example, it is prohibited to use preimplantation genetic diagnostics to choose the sex of an unborn child. But such a procedure is not prohibited in the States. But whether the Chinese are concerned about ethical issues or demographic reasons are more important for such legislative regulation is a big question.

But it is important that not only governments, but also ordinary people form their views on the boundaries of genetic interventions.  Otherwise, we risk finding ourselves in a situation of ignorance of the population about the basic principles of genetic technologies and the spread of prejudice. The recent wave of statements about the dangers of GMOs for humans is a vivid confirmation of this. One of the surveys in Kazan, for example, showed that almost half of the respondents believe that "any food products containing genes" should be withdrawn from sale and should not be imported or produced in the country. Obviously, there are genes in any living organism, so such research results are simply deplorable.  However, 15% of respondents honestly admitted that they do not imagine what GMOs are. Scientists, biomedics and just those who believe that technology makes our lives better, now we need to do everything so that human genetic editing does not face the same wave of unjustified panic, but becomes a truly effective tool in the fight against diseases.

Portal "Eternal youth" http://vechnayamolodost.ru  02.12.2016

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