Gene therapy must be carried out. Genetic diseases - treatment in Germany. Treatment of genetic diseases in children

Genetic diseases are diseases that arise in humans due to chromosomal mutations and defects in genes, that is, in the hereditary cellular apparatus. Damage to the genetic apparatus leads to serious and varied problems - hearing loss, visual impairment, delayed psycho-physical development, infertility and many other diseases.

The concept of chromosomes

Each cell of the body has a cell nucleus, the main part of which is made up of chromosomes. A set of 46 chromosomes is a karyotype. 22 pairs of chromosomes are autosomes, and the last 23 pair are sex chromosomes. These are the sex chromosomes that differentiate a man and a woman from each other.

Everyone knows that women have XX chromosomes, and men have XY chromosomes. When a new life arises, the mother passes on the X chromosome, and the father - either X or Y. It is with these chromosomes, or rather with their pathology, that genetic diseases are associated.

The gene can mutate. If it is recessive, then the mutation can be passed on from generation to generation without manifesting itself in any way. If the mutation is dominant, then it will definitely manifest itself, so it is advisable to protect your family by learning about the potential problem in time.

Genetic diseases are a problem in the modern world.

More and more hereditary pathologies are being discovered every year. More than 6,000 names of genetic diseases are already known; they are associated with both quantitative and qualitative changes in the genetic material. According to the World Health Organization, approximately 6% of children suffer from hereditary diseases.

The most unpleasant thing is that genetic diseases can appear only after several years. Parents rejoice at a healthy baby, not suspecting that their children are sick. For example, some hereditary diseases can manifest themselves at the age when the patient himself has children. And half of these children may be doomed if the parent carries a dominant pathological gene.

But sometimes it is enough to know that the child’s body is not able to absorb a certain element. If parents are warned about this in time, then in the future, simply avoiding products containing this component, you can protect the body from manifestations of a genetic disease.

Therefore, it is very important that when planning a pregnancy, a test for genetic diseases is done. If the test shows the likelihood of transmitting the mutated gene to the unborn child, then in German clinics they can carry out gene correction during artificial insemination. Tests can also be done during pregnancy.

In Germany, you can be offered innovative technologies of the latest diagnostic developments that can dispel all your doubts and suspicions. About 1,000 genetic diseases can be detected before a child is born.

Genetic diseases - what are the types?

We will look at two groups of genetic diseases (actually there are more)

1. Diseases with a genetic predisposition.

Such diseases can manifest themselves under the influence of external environmental factors and are very dependent on individual genetic predisposition. Some diseases may appear in older people, while others may appear unexpectedly and early. So, for example, a strong blow to the head can provoke epilepsy, taking an indigestible product can cause severe allergies, etc.

2. Diseases that develop in the presence of a dominant pathological gene.

Such genetic diseases are passed on from generation to generation. For example, muscular dystrophy, hemophilia, six-fingered, phenylketonuria.

Families at high risk of having a child with a genetic disease.

Which families first need to attend genetic consultations and identify the risk of hereditary diseases in their offspring?

1. Consanguineous marriages.

2. Infertility of unknown etiology.

3. Age of parents. It is considered a risk factor if the expectant mother is over 35 years old and the father is over 40 (according to some sources, over 45). With age, more and more damage appears in the reproductive cells, which increases the risk of having a baby with a hereditary pathology.

4. Hereditary family diseases, that is, similar diseases in two or more family members. There are diseases with pronounced symptoms and the parents have no doubt that this is a hereditary disease. But there are signs (microanomalies) that parents do not pay due attention to. For example, an unusual shape of the eyelids and ears, ptosis, coffee-colored spots on the skin, a strange smell of urine, sweat, etc.

5. Complicated obstetric history - stillbirth, more than one spontaneous miscarriage, missed pregnancies.

6. Parents are representatives of a small nationality or come from one small locality (in this case, there is a high probability of consanguineous marriages)

7. The impact of unfavorable household or professional factors on one of the parents (calcium deficiency, insufficient protein nutrition, work in a printing house, etc.)

8. Poor environmental conditions.

9. Use of drugs with teratogenic properties during pregnancy.

10. Diseases, especially viral etiology (rubella, chicken pox), suffered by a pregnant woman.

11. Unhealthy lifestyle. Constant stress, alcohol, smoking, drugs, poor nutrition can cause damage to genes, since the structure of chromosomes under the influence of unfavorable conditions can change throughout life.

Genetic diseases - what are the diagnostic methods?

In Germany, the diagnosis of genetic diseases is highly effective, since all known high-tech methods and absolutely all the capabilities of modern medicine (DNA analysis, DNA sequencing, genetic passport, etc.) are used to determine potential hereditary problems. Let's look at the most common ones.

1. Clinical and genealogical method.

This method is an important condition for high-quality diagnosis of a genetic disease. What does it include? First of all, a detailed interview with the patient. If there is a suspicion of a hereditary disease, then the survey concerns not only the parents themselves, but also all relatives, that is, complete and thorough information is collected about each family member. Subsequently, a pedigree is compiled indicating all the signs and diseases. This method ends with a genetic analysis, on the basis of which a correct diagnosis is made and optimal therapy is selected.

2. Cytogenetic method.

Thanks to this method, diseases that arise due to problems in the cell's chromosomes are determined. The cytogenetic method examines the internal structure and arrangement of chromosomes. This is a very simple technique - a scraping is taken from the mucous membrane of the inner surface of the cheek, then the scraping is examined under a microscope. This method is carried out with parents and family members. A type of cytogenetic method is molecular cytogenetic, which allows you to see the smallest changes in the structure of chromosomes.

3. Biochemical method.

This method, by examining the mother’s biological fluids (blood, saliva, sweat, urine, etc.), can determine hereditary diseases based on metabolic disorders. One of the most famous genetic diseases associated with metabolic disorders is albinism.

4. Molecular genetic method.

This is the most progressive method currently used to identify monogenic diseases. It is very accurate and detects pathology even in the nucleotide sequence. Thanks to this method, it is possible to determine a genetic predisposition to the development of oncology (cancer of the stomach, uterus, thyroid gland, prostate, leukemia, etc.) Therefore, it is especially indicated for persons whose close relatives suffered from endocrine, mental, oncological and vascular diseases.

In Germany, to diagnose genetic diseases, you will be offered the full range of cytogenetic, biochemical, molecular genetic studies, prenatal and postnatal diagnostics, plus neonatal screening of the newborn. Here you can take about 1,000 genetic tests that are approved for clinical use in the country.

Pregnancy and genetic diseases

Prenatal diagnosis provides great opportunities for identifying genetic diseases.

Prenatal diagnosis includes studies such as

• chorionic villus biopsy - analysis of fetal chorionic tissue at 7-9 weeks of pregnancy; a biopsy can be performed in two ways - through the cervix or by puncturing the anterior abdominal wall;
• amniocentesis - at 16-20 weeks of pregnancy, amniotic fluid is obtained through puncture of the anterior abdominal wall;
• Cordocentesis is one of the most important diagnostic methods, as it examines fetal blood obtained from the umbilical cord.

Screening methods such as triple test, fetal echocardiography, and determination of alpha-fetoprotein are also used in diagnosis.

Ultrasound imaging of the fetus in 3D and 4D dimensions can significantly reduce the birth of babies with developmental defects. All these methods have a low risk of side effects and do not adversely affect the course of pregnancy. If a genetic disease is detected during pregnancy, the doctor will suggest certain individual tactics for managing the pregnant woman. In the early stages of pregnancy, German clinics can offer gene correction. If gene correction is carried out in time in the embryonic period, then some genetic defects can be corrected.

Neonatal screening of a child in Germany

Neonatal newborn screening identifies the most common genetic diseases in an infant. Early diagnosis makes it possible to understand that a child is sick even before the first signs of illness appear. Thus, the following hereditary diseases can be identified - hypothyroidism, phenylketonuria, maple syrup disease, adrenogenital syndrome and others.

If these diseases are detected in time, the chance of curing them is quite high. High-quality neonatal screening is also one of the reasons why women fly to Germany to give birth to a child here.

Treatment of human genetic diseases in Germany

Until recently, genetic diseases were not treated; it was considered impossible, and therefore hopeless. Therefore, the diagnosis of a genetic disease was regarded as a death sentence, and at best, one could only count on symptomatic treatment. Now the situation has changed. Progress is noticeable, positive treatment results have appeared, and what’s more, science is constantly discovering new and effective ways to treat hereditary diseases. And although many hereditary diseases cannot be cured today, geneticists are optimistic about the future.

Treatment of genetic diseases is a very complex process. It is based on the same principles of influence as any other disease - etiological, pathogenetic and symptomatic. Let's look briefly at each.

1. Etiological principle of influence.

The etiological principle of influence is the most optimal, since treatment is aimed directly at the causes of the disease. This is achieved using methods of gene correction, isolating the damaged part of DNA, cloning it and introducing it into the body. At the moment, this task is very difficult, but for some diseases it is already feasible

2. Pathogenetic principle of influence.

Treatment is aimed at the mechanism of development of the disease, that is, it changes the physiological and biochemical processes in the body, eliminating defects caused by the pathological gene. As genetics develops, the pathogenetic principle of influence expands, and for different diseases, new ways and possibilities for correcting damaged links will be found every year.

3. Symptomatic principle of influence.

According to this principle, treatment of a genetic disease is aimed at relieving pain and other unpleasant phenomena and preventing further progression of the disease. Symptomatic treatment is always prescribed; it can be combined with other methods of treatment, or it can be an independent and sole treatment. This is the prescription of painkillers, sedatives, anticonvulsants and other medications. The pharmacological industry is now very developed, so the range of drugs used to treat (or rather, to alleviate the manifestations of) genetic diseases is very wide.

In addition to drug treatment, symptomatic treatment includes the use of physiotherapeutic procedures - massage, inhalations, electrotherapy, balneotherapy, etc.

Sometimes surgical treatment is used to correct deformities, both external and internal.

Geneticists in Germany already have extensive experience in treating genetic diseases. Depending on the manifestation of the disease and individual parameters, the following approaches are used:

• genetic nutrition;
• gene therapy,
• stem cell transplantation,
• organ and tissue transplantation,
• enzyme therapy,
• hormone and enzyme replacement therapy;
• hemosorption, plasmaphoresis, lymphosorption - cleansing the body with special preparations;
• surgery.

Of course, treatment of genetic diseases takes a long time and is not always successful. But the number of new approaches to therapy is growing every year, so doctors are optimistic.

Gene therapy

Doctors and scientists around the world place special hopes on gene therapy, thanks to which it is possible to introduce high-quality genetic material into the cells of a sick organism.

Gene correction consists of the following stages:

• obtaining genetic material (somatic cells) from the patient;
• introduction of a therapeutic gene into this material, which corrects the gene defect;
• cloning of corrected cells;
• introduction of new healthy cells into the patient’s body.

Gene correction requires great caution, since science does not yet have complete information about the functioning of the genetic apparatus.

List of genetic diseases that can be identified

There are many classifications of genetic diseases, they are arbitrary and differ in the principle of construction. Below we provide a list of the most common genetic and hereditary diseases:

• Gunther's disease;
• Canavan disease;
• Niemann-Pick disease;
• Tay-Sachs disease;
• Charcot-Marie disease;
• hemophilia;
• hypertrichosis;
• color blindness - insensitivity to color, color blindness is transmitted only with the female chromosome, but the disease affects only men;
• Capgras fallacy;
• Pelizaeus-Merzbacher leukodystrophy;
• Blashko lines;
• micropsia;
• cystic fibrosis;
• neurofibromatosis;
• heightened reflection;
• porphyria;
• progeria;
• spina bifida;
• Angelman syndrome;
• exploding head syndrome;
• blue skin syndrome;
• Down syndrome;
• living corpse syndrome;
• Joubert syndrome;
• stone man syndrome
• Klinefelter's syndrome;
• Klein-Levin syndrome;
• Martin-Bell syndrome;
• Marfan syndrome;
• Prader-Willi syndrome;
• Robin's syndrome;
• Stendhal's syndrome;
• Turner syndrome;
• elephantiasis;
• phenylketonuria.
• cicero and others.

In this section we will go into detail about each disease and tell you how some of them can be cured. But it is better to prevent genetic diseases than to treat them, especially since modern medicine does not know how to cure many diseases.

Genetic diseases are a group of diseases that are very heterogeneous in their clinical manifestations. The main external manifestations of genetic diseases:

• small head (microcephaly);
• microanomalies (“third eyelid”, short neck, unusually shaped ears, etc.)
• delayed physical and mental development;
• changes in genital organs;
• excessive muscle relaxation;
• change in the shape of the toes and hands;
• violation of psychological status, etc.

Genetic diseases - how to get advice in Germany?

Conversation in genetic consultation and prenatal diagnosis can prevent severe hereditary diseases transmitted at the gene level. The main goal of genetic counseling is to identify the degree of risk of a genetic disease in a newborn.

In order to receive quality consultation and advice on further actions, you need to be serious about communicating with your doctor. Before the consultation, you need to responsibly prepare for the conversation, remember the illnesses that your relatives suffered, describe all health problems and write down the main questions to which you would like to receive answers.

If the family already has a child with an anomaly, with congenital malformations, take his photographs. It is imperative to talk about spontaneous miscarriages, cases of stillbirth, and how the pregnancy went (is going).

A genetic consultation doctor will be able to calculate the risk of having a baby with a severe hereditary pathology (even in the future). When can we talk about a high risk of developing a genetic disease?

• a genetic risk of up to 5% is considered low;
• no more than 10% - slightly increased risk;
• from 10% to 20% - average risk;
• above 20% - high risk.

Doctors advise considering a risk of about or above 20% as a reason to terminate the pregnancy or (if one does not exist yet) as a contraindication to conception. But the final decision is made, of course, by the married couple.

The consultation may take place in several stages. When diagnosing a genetic disease in a woman, the doctor develops management tactics before pregnancy and, if necessary, during pregnancy. The doctor talks in detail about the course of the disease, life expectancy for this pathology, all the possibilities of modern therapy, the price component, and the prognosis of the disease. Sometimes gene correction during artificial insemination or during embryonic development allows one to avoid the manifestations of the disease. Every year, new methods of gene therapy and the prevention of hereditary diseases are being developed, so the chances of curing genetic pathology are constantly increasing.

In Germany, methods of combating gene mutations using stem cells are being actively introduced and are already being successfully applied, and new technologies are being considered for the treatment and diagnosis of genetic diseases.

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 a fairly high level of organ and tissue specificity. Such constructs are usually administered intravenously, intraperitoneally, subcutaneously or intramuscularly.

A number of special methods have been developed for "targeted delivery" of non-viral vectors. The simplest method of delivering the desired gene to cells in vivo is direct injection of genetic material into the 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 the respiratory tract 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, a 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 cause 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 the Food and Drug Administration (FDA) will consider continuing gene therapy experiments on a case-by-case basis, and only if there are no other treatments for 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 applications. 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.

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Establishing the location and sequence of the gene whose mutations cause specific diseases, as well as the mutation itself and modern methods of testing it, make 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 medication, diet, 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 the axons of nerve cells of the central nervous system and, as a result, 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 have complex clinical manifestations, and their traditional treatment is mainly symptomatic: to treat phenylketonuria, an alanine-free diet is prescribed, defective proteins are replaced with functional intravenous administration, and bone marrow or other organ transplantation is performed to compensate for lost functions. 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 the genetic defect that causes a 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, the toxic effect of which leads to the death of peripheral blood B and T lymphocytes 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. Gene therapy is being tested for a number of genetic diseases, including such severe ones as hemophilia, AIDS, various types of malignant neoplasms, cystic fibrosis, etc. At the moment, about 10 human diseases can 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 of 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 that affect 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 ineffective way is to inject 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 the DNA is subject to 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, in addition to the risk of a severe immune reaction, are the difficulties of long-term storage and functioning of therapeutic DNA in the patient's body, the multigenic nature of many diseases, making them difficult targets for gene therapy, and the risk of using viruses as vectors.

ON THE. Voinov, T.G. Volova

Gene therapy is one of the rapidly developing areas of medicine, which involves treating a person by introducing healthy genes into the body. Moreover, according to scientists, with the help of gene therapy it is possible to add a missing gene, correct or replace it, thereby improving the functioning of the body at the cellular level and normalizing the patient’s condition.

According to scientists, 200 million people on the planet are currently potential candidates for gene therapy, and this figure is steadily growing. And it is very gratifying that several thousand patients have already received treatment for incurable illnesses as part of ongoing trials.

In this article we will talk about what tasks gene therapy sets itself, what diseases can be treated with this method, and what problems scientists have to face.

Where is gene therapy used?

Gene therapy was originally conceived to combat severe inherited diseases such as Huntington's disease, cystic fibrosis and certain infectious diseases. However, the year 1990, when scientists managed to correct a defective gene and, by introducing it into the patient’s body, defeat cystic fibrosis, became truly revolutionary in the field of gene therapy. Millions of people around the world have received hope for the treatment of diseases that were previously considered incurable. And although such therapy is at the very beginning of its development, its potential is surprising even in the scientific world.

For example, in addition to cystic fibrosis, modern scientists have made progress in the fight against such hereditary pathologies as hemophilia, enzymopathy and immunodeficiency. Moreover, gene treatment makes it possible to fight some oncological diseases, as well as heart pathologies, diseases of the nervous system and even injuries, for example, nerve damage. Thus, gene therapy deals with extremely severe diseases that lead to early mortality and often have no other treatment other than gene therapy.

Principle of gene treatment

As an active substance, doctors use genetic information, or, to be more precise, molecules that are carriers of such information. Less commonly, RNA nucleic acids are used for this, and more often DNA cells are used.

Each such cell has a so-called “copier” - a mechanism by which it translates genetic information into proteins. A cell that has the correct gene and the photocopier works without failures is a healthy cell from the point of view of gene therapy. Each healthy cell has a whole library of original genes, which it uses for the correct and harmonious functioning of the entire organism. However, if for some reason an important gene is lost, it is not possible to restore such loss.

This becomes the cause of the development of serious genetic diseases, such as Duchenne muscular dystrophy (with it, the patient develops muscle paralysis, and in most cases he does not live to be 30 years old, dying from respiratory arrest). Or a less fatal situation. For example, a “breakdown” of a certain gene leads to the fact that the protein ceases to perform its functions. And this becomes the cause of the development of hemophilia.

In any of the listed cases, gene therapy comes to the rescue, the task of which is to deliver a normal copy of the gene to a diseased cell and place it in a cellular “copier”. In this case, the functioning of the cell will improve, and perhaps the functioning of the entire body will be restored, thanks to which the person will get rid of a serious illness and will be able to prolong his life.

What diseases can gene therapy treat?

How much does gene therapy really help a person? According to scientists, there are about 4,200 diseases in the world that arise as a result of malfunctioning genes. In this regard, the potential of this area of ​​medicine is simply incredible. However, what is much more important is what doctors have achieved so far. Of course, there are a lot of difficulties along this path, but today a number of local victories can be identified.

For example, modern scientists are developing approaches to treating coronary heart disease through genes. But this is an incredibly common disease that affects many more people than congenital pathologies. Ultimately, a person faced with coronary disease finds himself in a state where gene therapy can be his only salvation.

Moreover, today pathologies associated with damage to the central nervous system are treated with the help of genes. These are diseases such as amyotrophic lateral sclerosis, Alzheimer's disease or Parkinson's disease. Interestingly, to treat these ailments, viruses are used that tend to attack the nervous system. Thus, with the help of the herpes virus, cytokines and growth factors are delivered to the nervous system, slowing down the development of the disease. This is a striking example of how a pathogenic virus that usually causes disease is processed in the laboratory, stripped of disease-carrying proteins, and used as a cassette that delivers healing substances to the nerves and thereby acts for the benefit of health, prolonging human life.

Another serious hereditary disease is cholesterolemia, which causes the human body to be unable to regulate cholesterol, as a result of which fat accumulates in the body, and the risk of heart attacks and strokes increases. To cope with this problem, specialists remove part of the patient's liver and correct the damaged gene, stopping further accumulation of cholesterol in the body. The corrected gene is then placed into a neutralized hepatitis virus and sent back to the liver.

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There are positive developments in the fight against AIDS. It is no secret that AIDS is caused by the human immunodeficiency virus, which destroys the immune system and opens the door to deadly diseases in the body. Modern scientists already know how to change genes so that they stop weakening the immune system, and begin to strengthen it to counter the virus. Such genes are introduced through blood, through blood transfusion.

Gene therapy also works against cancer, in particular against skin cancer (melanoma). Treatment of such patients involves the introduction of genes with tumor necrosis factors, i.e. genes that contain antitumor proteins. Moreover, today trials are being conducted for the treatment of brain cancer, where sick patients are injected with a gene containing information to increase the sensitivity of malignant cells to the drugs used.

Gaucher disease is a severe hereditary disease that is caused by a mutation in a gene that suppresses the production of a special enzyme, glucocerebrosidase. In persons suffering from this incurable disease, the spleen and liver are enlarged, and as the disease progresses, bones begin to deteriorate. Scientists have already succeeded in experiments on introducing into the body of such patients a gene containing information on the production of this enzyme.

Here's another example. It is no secret that a blind person is deprived of the ability to perceive visual images for the rest of his life. One of the causes of congenital blindness is considered to be the so-called Leber atrophy, which, in fact, is a gene mutation. To date, scientists have restored visual abilities to 80 blind people using a modified adenovirus that delivered the “working” gene to the eye tissue. By the way, several years ago scientists managed to cure color blindness in experimental monkeys by introducing a healthy human gene into the retina of the animal’s eye. And more recently, such an operation allowed the first patients to cure color blindness.

Typically, the method of delivering genetic information using viruses is the most optimal, since viruses themselves find their targets in the body (the herpes virus will definitely find neurons, and the hepatitis virus will find the liver). However, this method of gene delivery has a significant drawback - viruses are immunogenic, which means that when they enter the body, they can be destroyed by the immune system before they have time to work, or even cause powerful immune responses from the body, only worsening the state of health.

There is another method of delivering gene material. It is a circular DNA molecule or plasmid. It spirals perfectly, becoming very compact, which allows scientists to “package” it into a chemical polymer and introduce it into a cell. Unlike a virus, a plasmid does not cause an immune response in the body. However, this method is less suitable, because after 14 days, the plasmid is removed from the cell and protein production stops. That is, in this way the gene must be introduced over a long period of time until the cell “recovers.”

Thus, modern scientists have two powerful methods for delivering genes to “sick” cells, and the use of viruses seems more preferable. In any case, the final decision on the choice of one method or another is made by the doctor, based on the reaction of the patient’s body.

Challenges facing gene therapy

A definite conclusion can be drawn that gene therapy is a poorly studied area of ​​medicine, which is associated with a large number of failures and side effects, and this is its huge drawback. However, there is also an ethical issue, because many scientists are categorically against interference in the genetic structure of the human body. That is why today there is an international ban on the use of germ cells, as well as pre-implantation germ cells, in gene therapy. This was done in order to prevent unwanted gene changes and mutations in our descendants.

Otherwise, gene therapy does not violate any ethical standards, because it is designed to fight serious and incurable diseases in which official medicine is simply powerless. And this is the most important advantage of gene treatment.
Take care of yourself!

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: the 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 the “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 several more years to comprehend the acquired knowledge and gradually begin to translate it into the field of practical application: first into diagnostic, and then into clinical practice.

Over the past 100 years, science has become quite good at dealing with the causative agents of various diseases, such as viruses and bacteria - 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 ($999–2500), it became increasingly affordable as the cost of sequencing 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: these are 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 tried to use this natural mechanism of resetting the biological clock to extend 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.