The gene therapy market has the potential to become the fastest growing market in the world in the next 10 years. The prospects that genetic manipulation opens motivate representatives of Big Pharma not only to conduct their own research, but also to actively buy up the most promising companies.
Pharmaceutical giant Novartis, apparently, can mark the beginning of the widespread introduction of gene therapy into global clinical practice: FDA food products and Medicines (Food and Drug Administration, FDA) has approved the use of gene therapy for patients aged 3 to 25 years suffering from acute lymphoblastic leukemia.
Treatment helps achieve remission, and in some cases even defeat the disease. The media have rightly dubbed this event a “new era of medicine” - humanity, with the help of genetic manipulation, is gradually coping with previously incurable diseases.
Let's look back at what led to the start of the "new era" and see where one of the most promising markets is heading.
How it all began
About 15 years ago, scientists managed to “read” the genome and finally gain access to the “source code” of the human body, which stores all the necessary data about it, and most importantly, controls its life and death. It took a few more years to comprehend the knowledge gained and gradually begin to translate it into the field practical application: first into diagnostic and then into clinical practice.
Over the past 100 years, managing pathogens various diseases, like viruses and bacteria, science has learned quite well - thanks to vaccines and antibiotics - but diseases caused by mutations in genes have long been considered incurable. Therefore, deciphering more than 3 billion nucleotide pairs has opened up truly unlimited prospects for the development of “medicine of the future” - primarily preventive genetic therapy, and, ideally, completely personalized medicine.
Market experts predict rapid growth in these areas: the cancer gene therapy market is projected to reach $4 billion by 2024, the gene therapy market as a whole is projected to reach $11 billion by 2025, and forecasts for all personalized medicine are even more optimistic: from $149 billion in 2020 to $2 .5 trillion by 2022.
The first fruits of deciphering the human genome were the improvement in the diagnosis of congenital diseases or predisposition to them (many will remember the case with the BRCA1 gene and Angelina Jolie). Against this background, the market for so-called “consumer genetics” began to develop rapidly - that by 2020 it will grow to $12 billion.
Genetic tests give the patient the opportunity to conduct an analysis and find “bad genes” in his body or, conversely, rejoice in their absence. Initially quite expensive pleasure($999–2500) became increasingly affordable as sequencing costs decreased. For example, the price of a comprehensive study offered today by one of the world market leaders, 23andMe, is $199. In Russia, prices are slightly higher: from 20,000 to 30,000 rubles.
In addition, targeted therapy is becoming a reality, which is especially important not only for hereditary diseases, but also for cardiovascular and infectious diseases, as well as oncology - the leading causes of death around the world. Genetic manipulation makes it possible to introduce “good” genes into a patient to compensate for the problems caused by the poor functioning of “bad” genes - for example, as in the case of hemophilia, and in the future they will also be able to “repair” or completely remove harmful genes - for example, those that cause neurodegenerative Huntington's disease. While gene therapy occupies a very modest place in the pharmaceutical market, its share is sure to grow steadily.
Of course, there are still many problems that need to be solved: this is the high risk of immune reactions, the high cost of therapy and, perhaps, even ethical issues associated with making changes to the human body at the genetic level. However, such manipulations are a chance for patients whose diseases are either considered incurable or cannot be effectively treated with existing drugs, as well as a new weapon in the fight against aging, giving humanity hope for healthy longevity at a completely different level, and the market - new ones, where more promising paths for development.
First victories
This program begins to operate from the moment of puberty and slowly but inexorably leads to death. Moreover, this is a fairly regulated process. Each species has a clear life limit that is allotted to it. In a mouse, for example, it is on average 2.5 years, in a person it is approximately 80 years. At the same time, there are other rodents that live several times or even an order of magnitude longer than mice - for example, squirrels or the famous naked mole rat.
The main question is whether aging can be turned off or at least slowed down. Perhaps a revolutionary technology that reverses cellular development, discovered by Shinya Yamanaka, a professor at the Institute of Advanced Medical Sciences at Kyoto University, will help answer this question: he found that inducing the co-expression of four transcription factors (Oct4, Sox2, Klf4 and c-Myc, and all together - OSKM, or Yamanaka factors), which are closely related to the main stages of the cell life cycle, turns somatic cells back into pluripotent ones. For this truly revolutionary discovery, Yamanaka received the Nobel Prize in 2012.
Using Yamanaka's breakthrough, a team of Salk Institute scientists led by Juan Carlos Izpisua Belmonte attempted to harness this natural reset mechanism. biological clock to prolong the life of adult animals. And I was not mistaken. Using Yamanaka factors, they were able to confirm the hypothesis about the possibility of rolling back the “epigenetic clock,” that is, cell rejuvenation, and increase the average life expectancy of rapidly aging mice by 33%-50% compared to various control groups.
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Establishing the location and sequence of a gene whose mutations cause specific diseases, as well as the mutation itself and modern methods its testing makes it possible to diagnose the disease in the neo- and even prenatal period of the body’s development. This makes it possible to mitigate the manifestation of a genetic defect with the help of drug treatment, diets, blood transfusions, etc.
However, this approach does not lead to correction of the defect itself and, as a rule, hereditary diseases are not cured. The situation is further complicated by the fact that a mutation in one gene can have very different effects on the body. If a gene mutation causes changes in the activity of the enzyme it encodes, this can lead to the accumulation of a toxic substrate or, conversely, to a deficiency of a compound necessary for normal cell functioning.
A well-known example of such a disease is phenylketonuria. It is caused by a mutation in the gene for the liver enzyme phenylalanine dehydroxylase, which catalyzes the conversion of phenylalanine to tyrosine. As a result, the level of endogenous phenylalanine in the blood increases, which causes improper formation of the myelin sheath around axons nerve cells central nervous system and, as a consequence, severe mental retardation.
If a mutation affects a structural protein gene, it can lead to serious disorders at the level of cells, tissues or organs. An example of such a disease is cystic fibrosis.
A deletion in the gene encoding a protein called the cystic fibrosis transporter results in defective protein synthesis (lacking phenylalanine 508) and impaired transport of chloride ions across cell membranes. One of the most harmful effects of this is that the mucus that lines and protects the lungs becomes abnormally thick. This makes it difficult to access lung cells and promotes the accumulation of harmful microorganisms. The cells lining the airways of the lungs die and are replaced by fibrous scar tissue (hence the name of the disease). As a result, the patient dies from respiratory failure.
Hereditary diseases are complex clinical manifestations, and their traditional treatment is mainly symptomatic: for the treatment of phenylketonuria, an alanine-free diet is prescribed, defective proteins are replaced with functional ones intravenous administration To compensate for lost functions, bone marrow or other organ transplantation is performed. All these measures are usually ineffective, expensive, time-consuming, and only a few patients live to old age. Therefore, the development of fundamentally new types of therapy is very important.
Gene therapy
Gene therapy is the genetic engineering of human somatic cells aimed at correcting a genetic defect, causing disease. Correction of a specific disease is carried out by introducing normally expressed genes into defective somatic cells. By the 1980s, when methods for obtaining individual genes were developed and eukaryotic expression vectors were created, gene transfer experiments in mice became routine, and the prospects for gene correction became real.In 1990, in the United States, Dr. W. French Andrson made the first attempt at gene therapy to treat severe combined immunodeficiency (SCID) in a three-year-old girl, Ashanti da Silva. This disease is caused by a mutation in the gene encoding adenosanadenylase (ADA). Deficiency of this enzyme contributes to the accumulation of adenosine and deoxyadenosine in the blood, toxic effect which leads to the death of B- and T-lymphocytes of peripheral blood and, as a consequence, immunodeficiency.
Children with this disease must be protected from any infections (kept in special sterile chambers), since any disease can be fatal. Four years after the start of treatment, the child showed expression of a normally functioning ADA and relief of SCID symptoms, allowing her to leave the sterile chamber and live a normal life.
Thus, the fundamental possibility of successful genetic therapy of somatic cells was demonstrated. Since the 90s. A number of gene therapies are being tested genetic diseases, including such severe ones as hemophilia, AIDS, different types malignant neoplasms, cystic fibrosis, etc. On this moment About 10 human diseases can already be cured using transgenesis.
The diversity of genetic diseases has led to the development of many gene therapy approaches. In this case, 2 main problems are solved: a means of delivering the therapeutic gene; a method for ensuring targeted delivery to cells intended for correction. To date, all approaches to gene therapy of somatic cells can be divided into two categories: ex vivo and in vivo therapy (Fig. 3.15).
Rice. 3.15. Scheme of gene therapy ex vivo (a) and in vivo (a)
Ex vivo gene therapy involves genetically correcting defective cells outside the body and then returning normally functioning cells back into the body.
In vivo gene therapy involves delivering a therapeutic gene directly into the cells of a specific patient tissue. Let's look at these approaches in more detail.
Ex vivo gene therapy includes the following steps:
1) obtaining defective cells from the patient and culturing them;
2) transfer of the desired gene into isolated cells using transfection of a therapeutic gene construct;
3) selection and expansion of genetically corrected cells;
4) transplantation or transfusion of these cells to the patient.
Using the patient's own cells ensures that they will not develop an immune response when they are returned. The procedure for transferring the gene construct must be efficient, and the normal gene must be stably maintained and continuously expressed.
The means of gene transfer created by nature itself are viruses. In order to obtain effective vectors for gene delivery, two groups of viruses are mainly used - adenoviruses and retroviruses (Fig. 3.16). In gene therapy, variants of genetically neutralized viruses are used.
Rice. 3.16. Viruses used to create therapeutic vectors
Let's consider the design and use of designs based on retroviruses. Let us recall that the genome of a retrovirus is represented by two identical single-stranded RNA molecules, each of which consists of six sections: two long terminal repeats (LTR) at the 5" and 3" ends, the non-coding sequence *P+, necessary for packaging the RNA into the viral particle, and three regions encoding the structural protein of the internal capsid (gag), reverse transcriptase (pol) and the envelope protein (env) (Fig. 3.17a).
Rice. 3.17. Genetic map of a typical retrovirus (a) and map of a retroviral vector (a)
Let us recall that the life cycle of a retrovirus includes the following stages:
1. Infection of target cells.
2. Synthesis of a DNA copy of the genome using its own reverse transcriptase.
3. Transport of viral DNA into the nucleus.
4. Incorporation of viral DNA into the host cell chromosome.
5. Transcription of mRNA from viral DNA under the control of a strong promoter localized in the 5"-LTR region.
6. Translation of Gag, Pol and Env proteins.
7. Formation of the viral capsid and packaging of two RNA chains and reverse transcriptase molecules.
8. Release of virions from the cell.
When obtaining a retroviral vector, the full-length DNA of the retrovirus is inserted into a plasmid, most of the gag gene and the entire pol and env genes are removed, and instead of them, a “therapeutic” T gene and, if necessary, a selective marker gene Rg with its own promoter are inserted (Fig. 3.17, b ). Transcription of the T gene will be controlled by the same strong promoter localized in the 5"-LTR region. Based on this scheme, various retroviral vectors and a maximum DNA insert size of approximately 8 thousand bp have been created.
The construct obtained in this way can itself be used for transformation, but its efficiency and subsequent integration into the host cell genome are extremely low. Therefore, a technique was developed for packaging full-length RNA of a retroviral vector into intact viral particles, which penetrate the cell with high frequency and are guaranteed to be integrated into the host genome. For this purpose, a so-called “packaging” cell line was created. In two different sections of the chromosomes of these cells, the retroviral genes gag and pol-env are embedded, deprived of the ability to pack due to the lack of the sequence + (84*+) (Fig. 3.18).
Rice. 3.18. Scheme for obtaining a packaged viral vector
That is, both of these fragments are transcribed, but empty capsids devoid of RNA are formed. When viral vector RNA is transfected into such cells, it is integrated into chromosomal DNA and transcribed to form full-length retroviral RNA, and under such conditions only the vector RNA is packaged in capsids (only it contains the + sequence). The resulting intact viral particles are used for efficient delivery of the retroviral vector to target cells.
Retroviruses actively infect only rapidly dividing cells. To transfer genes, they are treated with purified particles of the packaged retroviral vector or co-cultured with the cell line that produces them, and then selected to separate the target cells from the packaging cells.
Transduced cells are carefully checked for the level of synthesis of the therapeutic gene product, the absence of replication competent retroviruses, and the absence of changes in the ability of the cells to grow or function.
Bone marrow cells are the most suitable for gene therapy. This is due to the presence of totipotent embryonic stem cells, which can proliferate and differentiate into various types of cells - B and T lymphocytes, macrophages, erythrocytes, platelets and osteoclasts. It is these cells that are used to treat a number of hereditary diseases, including the already mentioned severe combined immunodeficiency, Gaucher disease, sickle cell anemia, thalassemia, osteoporosis, etc.
In addition to totipotent bone marrow stem cells, which are difficult to isolate and culture, stem cells from umbilical cord blood (the preferred use for gene therapy in newborns) are used, as well as liver cells - hepatocytes - to treat hypercholesterolemia.
In in vivo gene therapy, it is especially important to ensure delivery of the therapeutic gene to defective cells. Such targeted delivery can be provided by modified vectors created on the basis of viruses capable of infecting specific types cells. Consider the approach developed for the treatment of cystic fibrosis already mentioned above. Because the lungs are an open cavity, it is relatively easy to deliver therapeutic genes to them. The cloned version of the healthy gene was introduced into an inactivated adenovirus (Fig. 3.19). The specificity of this type of virus is that it infects the lining of the lungs, causing a cold.
Rice. 3.19. Scheme for obtaining a vector based on adenovirus
The virus thus constructed was tested by spraying it into the noses and lungs of experimental animals and then into human patients. In some cases, the introduction and expression of a healthy gene and the restoration of normal chloride ion transport were observed. It is possible that this approach (introducing a normal gene using nasal sprays) will be widely used in the near future to treat the symptoms of cystic fibrosis in the lungs.
In addition to retro- and adenoviruses, other types of viruses are also used in gene therapy experiments, for example the Herpes simplex virus. A special feature of this double-stranded (152 kb) DNA virus is its ability to specifically infect neurons. There are many known genetic diseases affecting the central and peripheral nervous system- tumors, metabolic disorders, neurodegenerative diseases (Alzheimer's disease, Parkinson's disease).
Herpes simplex virus type I (HSV) is a very suitable vector for the treatment of such diseases. The capsid of this virus fuses with the membrane of the neuron, and its DNA is transported to the nucleus. Several methods for transferring a therapeutic gene using HSV vectors have been proposed and successful tests have been carried out on experimental animals.
Viral vectors have several disadvantages: high cost, limited cloning capacity, and possible inflammatory response. Thus, in 1999, as a result of the development of an unusually strong immune response to the introduction of an adenoviral vector, an 18-year-old volunteer who took part in drug trials died. In 2002, two children in France developed a leukemia-like condition while being treated for immunodeficiency (by introducing therapeutic genes into stem cells using retroviruses).
Therefore, non-viral gene delivery systems are being developed. The simplest and most effective method- This is the injection of plasmid DNA into tissues. The second approach is bombarding tissues with gold microparticles (1-3 microns) conjugated to DNA. In this case, therapeutic genes are expressed in target tissues and their products - therapeutic proteins - enter the blood. The main disadvantage of this approach is the premature inactivation or destruction of these proteins by blood components.
DNA can be delivered by packaging it in an artificial lipid shell. The spherical liposome particles obtained in this way easily penetrate the cell membrane. Liposomes with a variety of properties have been created, but so far the efficiency of such delivery is low, since most of DNA undergoes lysosomal destruction. Also, to deliver a genetic construct, DNA conjugates are synthesized with various molecules that can ensure its safety, targeted delivery and penetration into the cell.
In recent years, intensive experiments have been carried out to create an artificial chromosome 47, which would allow the inclusion of a large amount of genetic material with a complete set of regulatory elements for one or more therapeutic genes. This would make it possible to use a genomic variant of a therapeutic gene and thereby ensure its stability and effective long-term expression. Experiments have shown that the creation of an artificial human chromosome containing therapeutic genes is quite possible, but it is not yet clear how to introduce such a huge molecule into the nucleus of a target cell.
The main challenges facing gene therapy, besides the risk of severe immune reaction, are the difficulties long-term storage and the functioning of therapeutic DNA in the patient’s body, the multigenic nature of many diseases, making them difficult targets for gene therapy, as well as the risk of using viruses as vectors.
ON THE. Voinov, T.G. Volova
Over its relatively short history, gene therapy has undergone ups and downs: sometimes scientists and practitioners saw it as almost a panacea, and then a period of disappointment and skepticism ensued...
Ideas about the possibility of introducing genes into the body for therapeutic purposes were expressed back in the early 60s of the last century, but real steps were taken only in the late 80s and were closely related to the international project to decipher the human genome.
In 1990, an attempt was made to gene therapy for severe, often incompatible with life, hereditary immunodeficiency caused by a defect in the gene encoding the synthesis of the enzyme adenosine deaminase. The study authors reported a clear therapeutic effect. And although over time a number of doubts arose about the durability of the effect obtained and its specific mechanisms, it was this work that served as a powerful impetus for the development of gene therapy and attracted multibillion-dollar investments.
Gene therapy is a medical approach based on the introduction of gene constructs into cells to treat various diseases. The desired effect is achieved either as a result of the expression of the introduced gene, or by suppressing the function of the defective gene. It should be emphasized that the goal of gene therapy is not to “treat” genes as such, but to treat various diseases with their help.
As a rule, a DNA fragment containing the necessary gene is used as a “drug”. It can simply be “naked DNA,” usually in combination with lipids, proteins, etc. But much more often, DNA is introduced as part of special genetic constructs (vectors) created on the basis of a variety of human and animal viruses using a number of genetic engineering manipulations. For example, genes necessary for its reproduction are removed from the virus. This, on the one hand, makes viral particles practically safe, and on the other, “frees up space” for genes intended for introduction into the body.
The fundamental point of gene therapy is the penetration of the gene construct into the cell (transfection), in the vast majority of cases - into its nucleus. It is important that the gene construct reaches exactly those cells that need to be “treated.” Therefore, the success of gene therapy largely depends on the choice of the optimal or at least satisfactory method of introducing gene constructs into the body.
With viral vectors, the situation is more or less predictable: they spread throughout the body and penetrate cells like their ancestor viruses, providing sufficient high level organ and tissue specificity. Such constructs are usually administered intravenously, intraperitoneally, subcutaneously or intramuscularly.
A number of non-viral vectors have been developed for "targeted delivery" special methods. The simplest method delivery of the desired gene to cells in vivo - direct injection of genetic material into tissue. The use of this method is limited: injections can only be made into the skin, thymus, striated muscles, and some solid tumors.
Another method of transgene delivery is ballistic transfection. It is based on “shelling” organs and tissues with microparticles of heavy metals (gold, tungsten) coated with DNA fragments. For “shelling” they use a special “gene gun”.
When treating lung diseases, it is possible to introduce genetic material into Airways in the form of an aerosol.
Cell transfection can also be carried out ex vivo: cells are isolated from the body, genetically manipulated, and then reintroduced into the patient’s body.
We treat: hereditary...
At the initial stage of development of gene therapy, its main objects were considered to be hereditary diseases caused by the absence or insufficient function of one gene, that is, monogenic ones. It was assumed that introducing a normally functioning gene to a patient would lead to a cure for the disease. Attempts have been made repeatedly to treat the “royal disease” - hemophilia, Duchenne muscular dystrophy, cystic fibrosis.
Today, gene therapy methods are being developed and tested for almost 30 monogenic human diseases. Meanwhile, there remain more questions than answers, and in most cases a real therapeutic effect has not been achieved. The reasons for this, first of all, are the body’s immune reaction, the gradual “attenuation” of the functions of the introduced gene, as well as the inability to achieve “targeted” integration of the transferred gene into chromosomal DNA.
Less than 10% of gene therapy studies are devoted to monogenic diseases, while the rest concern non-hereditary pathologies.
...and acquired
Acquired diseases are not associated with a congenital defect in the structure and function of genes. Their gene therapy is based on the principle that a “therapeutic gene” introduced into the body should lead to the synthesis of a protein that will either have a therapeutic effect or will help increase individual sensitivity to the effects of drugs.
Gene therapy can be used to prevent blood clots, restore the vascular system of the heart muscle after myocardial infarction, prevent and treat atherosclerosis, as well as in the fight against HIV infection and cancer. For example, a method of gene therapy for tumors, such as increasing the sensitivity of tumor cells to chemotherapy drugs, is being intensively developed; clinical trials are being conducted with the participation of patients with pleural mesothelioma, ovarian cancer, and glioblastoma. In 1999, a protocol for the treatment of prostate cancer was approved, safe doses of chemotherapy were selected, and a positive therapeutic effect was demonstrated.
Safety and Ethics
Carrying out genetic manipulations with the human body imposes special safety requirements: after all, any introduction of foreign genetic material into cells can have negative consequences. Uncontrolled integration of “new” genes into certain parts of the patient’s genome can lead to disruption of the function of “own” genes, which, in turn, can cause undesirable changes in the body, in particular the formation of cancerous tumors.
In addition, negative genetic changes can occur in somatic and germ cells. In the first case, we are talking about the fate of one person, where the risk associated with genetic correction is incomparably lower than the risk of death from an existing disease. When gene constructs are introduced into germ cells, undesirable changes in the genome can be transmitted to future generations. Therefore, it seems completely natural to want to ban experiments on genetic modification of germ cells not only for medical reasons, but also for ethical reasons.
A number of moral and ethical problems are associated with the development of approaches to gene intervention in the cells of a developing human embryo, that is, intrauterine gene therapy (in utero therapy). In the United States, the possibility of using gene therapy in utero is considered only for two severe genetic diseases: severe combined immunodeficiency caused by a defect in the adenosine deaminase enzyme gene, and homozygous beta thalassemia - severe hereditary disease, associated with the absence of all four globin genes or mutations in them. A number of gene constructs have already been developed and are being prepared for preliminary testing, the delivery of which to the body is expected to lead to compensation of genetic defects and elimination of the symptoms of these diseases. However, the risk of negative genetic consequences of such manipulations is quite high. Therefore, the ethics of intrauterine gene therapy also remains controversial.
In January of this year, experiments on gene therapy were again temporarily banned in the United States. The reason was dangerous complications that arose in two children after gene therapy for hereditary immunodeficiency. A few months ago in France, one of the children thought to be cured by gene therapy was diagnosed with a leukemia-like syndrome. Experts do not rule out that the use of retrovirus-based vectors during therapy may be the cause of the development of complications in children. Now representatives of the Food Control Agency and medicines(FDA) will consider continuing gene therapy experiments in individually, and only if there are no other methods of treating the disease.
Not a panacea, but a prospect
It cannot be denied that the actual success of gene therapy in treating specific patients is quite modest, and the approach itself is still at the stage of data accumulation and technology development. Gene therapy has not and, obviously, will never become a panacea. The body's regulatory systems are so complex and so little studied that simply introducing a gene in most cases does not produce the necessary therapeutic effect.
However, despite all this, the promise of gene therapy can hardly be overestimated. There is every reason to hope that progress in the field of molecular genetics and genetic engineering technologies will lead to undoubted success in the treatment of human diseases using genes. And, in the end, gene therapy will rightfully take its place in practical medicine.
It appears that gene therapy may have some unexpected uses. According to scientists, the Olympic Games will be held in 2012, where transgenic super athletes will perform. “DNA doping” will provide undoubted advantages
in developing strength, endurance and speed. There is no doubt that in the conditions of fierce competition in sports there will be athletes who are ready for genetic modification, even taking into account the possible risks associated with the use of new technology.
Human gene therapy, in a broad sense, involves introducing a functionally active gene(s) into cells to correct a genetic defect. There are two possible ways to treat hereditary diseases. In the first case, somatic cells (cells other than germ cells) are subjected to genetic transformation. In this case, correction of a genetic defect is limited to a specific organ or tissue. In the second case, the genotype of germline cells (sperm or eggs) or fertilized eggs (zygotes) is changed so that all the cells of the individual that develops from them have the “corrected” genes. Through gene therapy using germline cells, genetic changes are passed on from generation to generation.
Somatic cell gene therapy policy.
In 1980, representatives of the Catholic, Protestant and Jewish communities in the United States wrote an open letter to the President outlining their views on the use of genetic engineering in relation to humans. A Presidential Commission and a Congressional Commission were created to evaluate the ethical and social aspects of this problem. These were very important initiatives, since in the United States the enactment of programs affecting the public interest is often carried out on the basis of the recommendations of such commissions. The final conclusions of both commissions drew a clear distinction between gene therapy of somatic cells and gene therapy of germline cells. Gene therapy of somatic cells has been classified as a standard method of medical intervention in the body, similar to organ transplantation. In contrast, germline cell gene therapy has been considered technologically too difficult and ethically too challenging to implement immediately. It was concluded that there is a need to develop clear rules governing research in the field of gene therapy of somatic cells; the development of similar documents in relation to gene therapy of germline cells was considered premature. In order to stop all illegal activities, it was decided to stop all experiments in the field of gene therapy of germline cells.
By 1985, they had developed a document entitled “Regulations on the preparation and submission of applications for experiments in the field of gene therapy of somatic cells.” It contained all the information about what data must be submitted in an application for permission to test somatic cell gene therapy in humans. The basis was taken from the rules governing laboratory research with recombinant DNA; they have only been adapted for biomedical purposes.
Biomedical legislation was revised and expanded in the 1970s. in response to the 1972 release of the results of a 40-year experiment conducted by the National Health Service in Alabama on a group of 400 illiterate African Americans with syphilis. The experiment was carried out in order to study the natural development of this sexually transmitted disease; no treatment was carried out. The news of such a horrendous experience on uninformed people shocked many in the United States. Congress immediately stopped the experiment and passed a law prohibiting such research from ever being conducted again.
Among the questions addressed to persons who applied for permission to experiment in the field of gene therapy of somatic cells were the following:
- 1. What is the disease that is supposed to be treated?
- 2. How serious is it?
- 3. Are there alternative treatments?
- 4. How dangerous is the proposed treatment for patients?
- 5. What is the probability of treatment success?
- 6. How will patients be selected for clinical trials?
- 7. Will this selection be unbiased and representative?
- 8. How will patients be informed about the tests?
- 9. What kind of information should they be given?
- 10. How will their consent be obtained?
- 11. How will the confidentiality of information about patients and research be guaranteed?
When gene therapy experiments first began, most applications for clinical trials were first reviewed by the Ethics Committee of the institution where the research was to be carried out before being forwarded to the Human Gene Therapy Subcommittee. The latter assessed applications from the point of view of their scientific and medical significance, compliance with current rules, and the persuasiveness of the arguments. If the application was rejected, it was returned with the necessary comments. The authors of the proposal could review the proposal and rework it. If an application was approved, the Gene Therapy Subcommittee discussed it in public discussions using the same criteria. After approval of the application at this level, the director of the Subcommittee approved it and signed the authorization for clinical trials, without which they could not begin. In this latter case, special attention was paid to the method of obtaining the product, methods of qualitative control of its purity, and what preclinical tests were conducted to ensure the safety of the product.
But as the number of applications increased over time, and gene therapy became, in the words of one commentator, “the winning ticket in medicine,” the original application approval process was considered unnecessarily time-consuming and redundant. Accordingly, after 1997, the Gene Therapy Subcommittee was no longer one of the agencies overseeing human gene therapy research. If the Subcommittee exists, it will most likely provide forums to discuss ethical issues related to human gene therapy. In the meantime, the requirement that all gene therapy applications be discussed publicly has been lifted. The agency responsible for monitoring the production and use of biological products conducts all necessary assessments confidentially to ensure that the developers' proprietary rights are respected. Human gene therapy is currently considered safe medical procedure, although not particularly effective. Previously expressed concerns have dissipated, and it has become one of the main new approaches to the treatment of human diseases.
Most experts consider the approval process for human somatic cell gene therapy trials in the United States to be quite adequate; it guarantees the impartial selection of patients and their awareness, as well as the implementation of all manipulations properly, without causing harm to both specific patients and the human population as a whole. Other countries are also currently developing regulations for gene therapy trials. In the US this was done by carefully weighing each proposal. As Dr. Walters, one of the participants in the Gene Therapy Subcommittee hearings in January 1989, said: "I know of no other biomedical science or technology that has been subjected to such extensive scrutiny as gene therapy."
Accumulation of defective genes in future generations.
There is an opinion that the treatment of genetic diseases using gene therapy of somatic cells will inevitably lead to a deterioration in the gene pool of the human population. It is based on the idea that the frequency of a defective gene in a population will increase from generation to generation, since gene therapy will promote the transmission of mutant genes to the next generation from those people who were previously unable to produce offspring or could not survive to adulthood. However, this hypothesis turned out to be incorrect. According to population genetics, for a significant increase in the frequency of a harmful or lethal gene as a result effective treatment it takes thousands of years. Thus, if a rare genetic disease occurs in 1 in 100,000 live births, it will take approximately 2,000 years after the introduction of effective gene therapy before the incidence of the disease doubles to 1 in 50,000.
In addition to the fact that the frequency of the lethal gene almost does not increase from generation to generation, as a result long-term treatment everyone who needs it, the genotype of individual individuals also remains unchanged. This point can be illustrated with an example from the history of evolution. Primates, including humans, are unable to synthesize vital important vitamin C, they must obtain it from external sources. Thus, we can say that we are all genetically defective in the gene for this vital substance. In contrast, amphibians, reptiles, birds, and non-primate mammals synthesize vitamin C. Yet the genetic defect that causes the inability to biosynthesize vitamin C did not “prevent” the successful evolution of primates for more than millions of years. Likewise, correcting other genetic defects will not lead to a significant accumulation of “unhealthy” genes in future generations.
Gene therapy of germline cells.
Experiments in the field of gene therapy of human germline cells are now strictly prohibited, but it must be recognized that some genetic diseases can only be cured in this way. The methodology for gene therapy of human germline cells has not yet been sufficiently developed. However, there is no doubt that with the development of methods of genetic manipulation in animals and diagnostic testing preimplantation embryos will fill this gap. Moreover, as somatic cell gene therapy becomes more routine, this will affect people's attitudes toward human germline gene therapy, and over time there will be a need to test it. One can only hope that by that time all the problems associated with the consequences of the practical use of gene therapy for human germline cells, including social and biological ones, will be resolved.
Human gene therapy is thought to help treat serious illnesses. Indeed, it can provide correction for a number of physical and mental disorders, although it remains unclear whether society will find such use of gene therapy acceptable. Like any other new medical direction, gene therapy of human germline cells raises numerous questions, namely:
- 1. What is the cost of developing and implementing gene therapy methods for human germline cells?
- 2. Should the government set medical research priorities?
- 3. Will the priority development of gene therapy for germline cells lead to the curtailment of work on finding other methods of treatment?
- 4. Will it be possible to reach all patients who need it?
- 5. Will an individual or company be able to obtain exclusive rights to treat specific diseases using gene therapy?
Human cloning.
Public interest in the possibility of human cloning arose in the 1960s, after corresponding experiments were carried out on frogs and toads. These studies showed that the nucleus of a fertilized egg can be replaced with the nucleus of an undifferentiated cell, and the embryo will develop normally. Thus, in principle, it is possible to isolate nuclei from undifferentiated cells of an organism, introduce them into fertilized eggs of the same organism, and produce offspring with the same genotype as the parent. In other words, each of the descendant organisms can be considered a genetic clone of the original donor organism. In the 1960s it seemed that, despite the lack of technical capabilities, it was not difficult to extrapolate the results of frog cloning to humans. Many articles on this topic appeared in the press, and even science fiction works were written. One of the stories was about the cloning of the treacherously assassinated US President John F. Kennedy, but a more popular topic was the cloning of villains. The works about human cloning were not only implausible, but also promoted the erroneous and very dangerous idea that a person’s personality traits, character and other qualities are determined solely by his genotype. In fact, a person as a personality is formed under the influence of both his genes and environmental conditions, in particular cultural traditions. For example, the malicious racism that Hitler preached is an acquired behavioral quality that is not determined by any one gene or their combination. In another environment with different cultural characteristics, the “cloned Hitler” would not necessarily have formed into a person similar to the real Hitler. Likewise, a “clone of Mother Teresa” would not necessarily “make” a woman who dedicated her life to helping the poor and sick in Calcutta.
As methods of mammalian reproductive biology developed and the creation of various transgenic animals, it became increasingly clear that human cloning was a matter of the not-too-distant future. The speculation became reality in 1997, when a sheep named Dolly was cloned. For this purpose, the nucleus of a differentiated cell from a donor sheep was used. The methodological approach that was used to “create” Dolly is, in principle, suitable for obtaining clones of any mammals, including humans. And even if it doesn't work out well in other mammal species, it probably won't take too much experimentation to develop a suitable method. As a result, human cloning will immediately become the subject of any discussion involving ethical problems of genetics and biological medicine.
Without a doubt, human cloning is a complex and controversial issue. For some, the very idea of creating a copy of an already existing individual through experimental manipulation seems unacceptable. Others believe that a cloned individual is the same as an identical twin, despite the age difference, and therefore cloning is not inherently malicious, although perhaps not entirely necessary. Cloning can have positive medical and social effects that justify its implementation in exceptional cases. For example, it may be vital for the parents of a sick child. Liability for human cloning experiments is regulated by law in many countries, and all research related to human cloning is prohibited. Such restrictions are enough to exclude the possibility of human cloning. However, the question of the inevitability of human cloning will certainly arise.
Duchenne muscular dystrophy is one of the rare, but still relatively common genetic diseases. The disease is diagnosed at the age of three to five, usually in boys, at first manifesting itself only in difficult movements; by the age of ten, a person suffering from such muscular dystrophy can no longer walk, and by the age of 20–22 his life ends. It is caused by a mutation in the dystrophin gene, which is located on the X chromosome. It encodes a membrane spanning protein muscle cell with contractile fibers. Functionally, it is a kind of spring that ensures smooth contraction and integrity of the cell membrane. Mutations in the gene lead to dystrophy of skeletal muscle tissue, diaphragm and heart. Treatment of the disease is palliative and can only slightly alleviate suffering. However, with the development of genetic engineering, there is light at the end of the tunnel.
About war and peace
Gene therapy is the delivery of nucleic acid-based constructs into cells to treat genetic diseases. With the help of such therapy, it is possible to correct a genetic problem at the DNA and RNA level, changing the process of expression of the desired protein. For example, DNA with a corrected sequence can be delivered into a cell, with which a functional protein is synthesized. Or, conversely, it is possible to remove certain genetic sequences, which will also help reduce the harmful effects of the mutation. In theory, this is simple, but in practice, gene therapy is based on the most complex technologies for working with microscopic objects and represents a set of advanced know-how in the field of molecular biology.
Injecting DNA into the pronucleus of the zygote is one of the earliest and most traditional technologies for creating transgenes. The injection is performed manually using ultra-fine needles under a microscope with 400x magnification.
“The dystrophin gene, mutations of which give rise to Duchenne muscular dystrophy, is huge,” says Vadim Zhernovkov, development director of the biotechnology company Marlin Biotech, candidate of biological sciences. “It includes 2.5 million nucleotide pairs, which could be compared to the number of letters in the novel War and Peace.” And let’s imagine that we have torn out several important pages from the epic. If these pages describe significant events, then understanding the book would already be difficult. But with the gene everything is more complicated. It would not be difficult to find another copy of War and Peace, and then the missing pages could be read. But the dystrophin gene is located on the X chromosome, and in men there is only one. Thus, only one copy of the gene is stored in the sex chromosomes of boys at birth. There is nowhere to get another one.
Finally, when synthesizing proteins from RNA, maintaining the reading frame is important. The reading frame determines which group of three nucleotides is read as a codon, corresponding to one amino acid in a protein. If a DNA fragment that is not a multiple of three nucleotides is deleted in a gene, the reading frame shifts—the encoding changes. This could be compared to the situation when, after torn out pages in the entire remaining book, all letters are replaced by the next ones in the alphabet. The result will be abracadabra. The same thing happens with improperly synthesized protein.”
Biomolecular patch
One of effective methods gene therapy to restore normal protein synthesis - exon skipping using short nucleotide sequences. Marlin Biotech has already developed the technology for working with the dystrophin gene using this method. As is known, in the process of transcription (RNA synthesis), the so-called pre-template RNA is first formed, which contains both protein-coding regions (exons) and non-coding regions (introns). Next, the splicing process begins, during which introns and exons are separated and a “mature” RNA is formed, consisting only of exons. At this moment, some exons can be blocked, “sealed” with the help of special molecules. As a result, the mature RNA will not contain those coding regions that we would prefer to get rid of, and thus the reading frame will be restored and the protein will be synthesized.
“We have debugged this technology in vitro,” says Vadim Zhernovkov, that is, on cell cultures grown from cells of patients with Duchenne muscular dystrophy. But individual cells are not an organism. By invading cellular processes, we must observe the consequences live, but it is not possible to involve people in testing due to various reasons- from ethical to organizational. Therefore, there was a need to obtain a laboratory animal model of Duchenne muscular dystrophy with certain mutations.”
How to inject a microcosm
Transgenic animals are animals obtained in the laboratory in which changes have been purposefully and deliberately made to their genome. Back in the 70s of the last century, it became clear that the creation of transgenes is the most important method studies of gene and protein functions. One of the earliest methods of obtaining a completely genetically modified organism was the injection of DNA into the pronucleus ("precursor of the nucleus") of zygotes of fertilized eggs. This is logical, since it is easiest to modify an animal’s genome at the very beginning of its development.
The diagram shows the CRISPR/Cas9 process, which involves a subgenomic RNA (sgRNA), a region of it that acts as guide RNA, and a Cas9 nuclease protein that cuts both strands of genomic DNA at the location specified by the guide RNA.
Injection into the nucleus of a zygote is a very non-trivial procedure, because we are talking about a microscale. The mouse egg has a diameter of 100 microns, and the pronucleus is 20 microns. The operation takes place under a microscope with 400x magnification, but the injection is the most handmade. Of course, for the “injection”, not a traditional syringe is used, but a special glass needle with a hollow channel inside, into which the gene material is collected. One end of it can be held in your hand, and the other - ultra-thin and sharp - is practically invisible to the naked eye. Of course, such a fragile structure made of borosilicate glass cannot be stored for a long time, so the laboratory has at its disposal a set of blanks that are drawn out on a special machine immediately before work. A special system of contrast visualization of the cell without staining is used - intervention in the pronucleus is in itself traumatic and is a risk factor for the survival of the cell. Paint would be another such factor. Fortunately, eggs are quite tenacious, but the number of zygotes that give rise to transgenic animals is only a few percent of the total. total number eggs into which DNA has been injected.
The next stage is surgical. An operation is being performed to transplant microinjected zygotes into the oviduct of the recipient mouse, which will become a surrogate mother for future transgenes. Next, the laboratory animal naturally goes through a pregnancy cycle, and offspring are born. Typically, a litter contains about 20% of transgenic mice, which also indicates the imperfection of the method, since there is a large element of randomness in it. When injected, the researcher cannot control exactly how the introduced DNA fragments will be integrated into the genome of the future organism. There is a high probability of such combinations that will lead to the death of the animal at the embryonic stage. Nevertheless, the method works and is quite suitable for a number of scientific purposes.
The development of transgenic technologies makes it possible to produce animal proteins that are in demand by the pharmaceutical industry. These proteins are extracted from the milk of transgenic goats and cows. There are also technologies for obtaining specific proteins from chicken eggs.
DNA scissors
But there is a more effective way based on targeted genome editing using CRISPR/Cas9 technology. “Today, molecular biology is somewhat similar to the era of long-distance sea expeditions under sail,” says Vadim Zhernovkov. — Almost every year significant discoveries occur in this science that can change our lives. For example, several years ago microbiologists discovered that a species of bacteria that had been studied for a long time was immune to viral infections. As a result further research It turned out that bacterial DNA contains special loci (CRISPR), from which RNA fragments are synthesized that can complementarily bind to the nucleic acids of foreign elements, for example, DNA or RNA viruses. The Cas9 protein, which is a nuclease enzyme, binds to such RNA. RNA serves as a guide for Cas9, marking a specific section of DNA where the nuclease makes a cut. About three to five years ago, the first scientific papers appeared in which CRISPR/Cas9 technology was developed for genome editing.”
Transgenic mice allow the creation of living models of severe human genetic diseases. People should be grateful to these tiny creatures.
Compared to the method of introducing a construct for random insertion, the new method allows you to select elements of the CRISPR/Cas9 system in such a way as to precisely target RNA guides to the desired regions of the genome and achieve targeted deletion or insertion of the desired DNA sequence. This method is also subject to errors (guide RNA sometimes binds to the wrong site where it is targeted), but when using CRISPR/Cas9, the efficiency of creating transgenes is already about 80%. “This method has broad prospects, not only for creating transgenes, but also in other areas, in particular in gene therapy,” says Vadim Zhernovkov. “However, the technology is only at the beginning of its journey, and it is quite difficult to imagine that in the near future people’s gene code will be corrected using CRISPR/Cas9. While there is a possibility of error, there is also a danger that a person will lose some important coding part of the genome.”
Milk medicine
The Russian company Marlin Biotech has managed to create a transgenic mouse in which the mutation leading to Duchenne muscular dystrophy is completely reproduced, and the next stage will be testing gene therapy technologies. However, the creation of models of human genetic diseases based on laboratory animals is not the only possible use of transgenes. Thus, in Russia and Western laboratories, work is underway in the field of biotechnology, making it possible to obtain medicinal proteins of animal origin that are important for the pharmaceutical industry. Cows or goats can act as producers, in which the cellular apparatus for producing proteins contained in milk can be changed. It is possible to extract medicinal protein from milk, which is not obtained chemically, but with the help of a natural mechanism, which will increase the effectiveness of the medicine. Currently, technologies have been developed for the production of medicinal proteins such as human lactoferrin, prourokinase, lysozyme, atrine, antithrombin and others.
