Types of gene therapy compensation for genetic defects. Gene therapy and medicine of the 21st century. Not a panacea, but perspective

Introduction

Every year, more and more articles appear in scientific journals about medical clinical research, in which, one way or another, a treatment based on the introduction of various genes - gene therapy - was used. This direction grew out of such well-developing branches of biology as molecular genetics and biotechnology.

Often, when conventional (conservative) methods have already been tried, it is gene therapy that can help patients survive and even fully recover. For example, this applies to hereditary monogenic diseases, that is, those that are caused by a defect in a single gene, as well as many others. Or, for example, gene therapy can help out and save a limb for those patients who have a narrowed vascular lumen in the lower extremities and, as a result, develop persistent ischemia of the surrounding tissues, that is, these tissues experience a strong lack of nutrients and oxygen, which are normally carried by the blood through the body. It is often impossible to treat such patients with surgical manipulations and medications, but if the cells were locally forced to throw out more protein factors that would affect the formation and germination of new vessels, then ischemia would become much less pronounced and the patient's life would become much easier.

Gene therapy today it can be defined as the treatment of diseases by introducing genes into the cells of patients with the aim of targeting gene defects or imparting new functions to cells. The first clinical trials of gene therapy methods were undertaken quite recently - May 22, 1989 in order to diagnose cancer. The first hereditary disease for which the methods of gene therapy were applied was hereditary immunodeficiency.

Every year the number of successfully conducted clinical trials for the treatment of various diseases using gene therapy is growing, and by January 2014 it had reached 2 thousand.

At the same time, in modern research on gene therapy, it must be borne in mind that the consequences of manipulating genes or "shuffled" (recombinant) DNA in vivo(Latin literally "live") have not been studied enough. In countries with the most advanced level of research in this area, especially in the United States, medical protocols using sense DNA sequences are subject to mandatory review by the appropriate committees and commissions. In the United States, these are the Recombinant DNA Advisory Committee (RAC) and the Food and Drug Administration (FDA), with the subsequent mandatory approval of the project by the director of the National Institutes of Health.

So, we decided that this treatment is based on the fact that if some body tissues lack some individual protein factors, then this can be corrected by introducing into these tissues the corresponding genes encoding proteins, and everything will become more or less wonderful. The proteins themselves cannot be injected, because our body will immediately react with a non-weak immune reaction, and the duration of the action would be insufficient. Now it is necessary to decide on the method of gene delivery into cells.

Transfection cells

To begin with, it is worth introducing definitions of some terms.

Gene transport is carried out thanks to vector is a DNA molecule used as a "vehicle" for the artificial transfer of genetic information into a cell. There are many types of vectors: plasmid, viral, as well as cosmids, phasmids, artificial chromosomes, etc. It is fundamentally important that vectors (in particular, plasmid ones) have their characteristic properties:

1. Replication origin (ori)- the sequence of nucleotides from which DNA doubling begins. If vector DNA cannot be duplicated (replicated), then the required therapeutic effect will not be achieved, because it is simply quickly cleaved by intracellular nuclease enzymes, and due to the lack of matrices, much fewer protein molecules will be formed as a result. It should be noted that these points are specific for each biological species, that is, if vector DNA is supposed to be obtained by its reproduction in a bacterial culture (and not just by chemical synthesis, which is usually much more expensive), then two points of replication start will be required separately - for humans and for bacteria;

2. Restriction sites- specific short sequences (usually palindromic), which are recognized by special enzymes (restriction endonucleases) and cut by them in a certain way - with the formation of "sticky ends" (Fig. 1).

Fig.1 Formation of "sticky ends" with the participation of restriction enzymes

These sites are required in order to ligate vector DNA (which is, in fact, a "blank") with the desired therapeutic genes into a single molecule. Such a molecule stitched from two or more parts is called "recombinant";

3. It is clear that we would like to get millions of copies of the recombinant DNA molecule. Again, if we are dealing with a culture of bacterial cells, then this DNA must be isolated. The problem is that not all bacteria will swallow the molecule we need, some will not. In order to distinguish between these two groups, they insert into the vector DNA selective markers- areas of resistance to certain chemicals; now if these same substances are added to the environment, then only those that are resistant to them will survive, and the rest will die.

All these three components can be observed in the very first artificially synthesized plasmid (Fig. 2).

Fig. 2

The very process of introducing a plasmid vector into certain cells is called transfection... A plasmid is a fairly short and usually circular DNA molecule that resides in the cytoplasm of a bacterial cell. Plasmids are not associated with the bacterial chromosome, they can replicate independently of it, they can be released by the bacteria into the environment, or, conversely, be absorbed (absorption process - transformation). With the help of plasmids, bacteria can exchange genetic information, for example, transfer resistance to certain antibiotics.

Plasmids exist naturally in bacteria. But no one can prevent a researcher from artificially synthesizing a plasmid that will have the properties he needs, to sew an insert gene into it and introduce it into a cell. Different inserts can be sewn into the same plasmid .

Gene therapy methods

There are two main approaches, which differ in the nature of the target cells:

1. Fetal, in which foreign DNA is introduced into the zygote (fertilized egg) or embryo at an early stage of development; at the same time, it is expected that the introduced material will enter all the cells of the recipient (and even into the germ cells, thereby ensuring transmission to the next generation). In our country, it is actually prohibited;

2. Somatic, in which the genetic material is introduced into the non-sex cells already born, and it is not transferred to the reproductive cells.

Gene therapy in vivo based on the direct introduction of cloned (multiplied) and in a certain way packed DNA sequences into certain tissues of the patient. The introduction of genes with the help of aerosol or injectable vaccines seems to be especially promising for the treatment of gene diseases in vivo. Aerosol gene therapy is usually developed for the treatment of pulmonary diseases (cystic fibrosis, lung cancer).

Many stages precede the development of a gene therapy program. This is a thorough analysis of the tissue-specific expression of the corresponding gene (i.e., the synthesis of a certain protein on the gene matrix in a certain tissue), and the identification of the primary biochemical defect, and the study of the structure, function and intracellular distribution of its protein product, as well as biochemical analysis of the pathological process. All these data are taken into account when drawing up an appropriate medical protocol.

It is important that when drawing up schemes for gene correction, the efficiency of transfection, the degree of correction of the primary biochemical defect in cell culture conditions ( in vitro,"in a test tube") and, what is especially important, in vivo on animals - biological models. Only then can the program of clinical trials be started. .

Direct delivery and cellular carriers of therapeutic genes

There are many methods for introducing foreign DNA into a eukaryotic cell: some depend on physical processing (electroporation, magnetofection, etc.), others on the use of chemical materials or biological particles (for example, viruses) that are used as carriers. It should be noted right away that chemical and physical methods are usually combined (for example, electroporation + enveloping DNA with liposomes)

Direct methods

1. Transfection on a chemical basis can be classified into several types: using a substance cyclodextrin, polymers, liposomes or nanoparticles (with or without chemical or viral functionalization, ie surface modification).
a) One of the cheapest methods is to use calcium phosphate. It increases the efficiency of DNA incorporation into cells by 10-100 times. DNA forms a strong complex with calcium, which ensures its efficient absorption. The disadvantage is that the nucleus reaches only about 1-10% of the DNA. Method used in vitro for transferring DNA into human cells (Fig. 3);

Fig. 3

b) The use of highly branched organic molecules - dendrimer, for binding DNA and transferring it into the cell (Fig. 4);

Fig. 4

c) A very effective method for transfection of DNA is its introduction through liposomes - small, membrane-surrounded bodies that can fuse with the cellular cytoplasmic membrane (CPM), which is a double layer of lipids. For eukaryotic cells, transfection is performed more efficiently using cationic liposomes, because the cells are more sensitive to them. The process has its own name - lipofection. This method is considered one of the safest today. Liposomes are non-toxic and non-immunogenic. However, the efficiency of gene transfer using liposomes is limited, since the DNA introduced by them into cells is usually immediately taken up by lysosomes and destroyed. The introduction of DNA into human cells with the help of liposomes is today the main thing in therapy. in vivo(fig. 5);

Fig. 5

d) Another method is the use of cationic polymers such as diethylaminoethyl-dextran or polyethyleneimine. Negatively charged DNA molecules bind to positively charged polycations, and this complex further enters the cell through endocytosis. DEAE-dextran alters the physical properties of the plasma membrane and stimulates the absorption of this complex by the cell. The main disadvantage of the method is that DEAE-dextran is toxic in high concentrations. The method has not become widespread in gene therapy;

e) With the help of histones and other nuclear proteins. These proteins, which contain many positively charged amino acids (Lys, Arg), naturally help to compactly pack a long DNA chain into a relatively small cell nucleus.

2. Physical methods:

a) Electroporation is a very popular method; an instant increase in membrane permeability is achieved due to the fact that cells are exposed to short exposures of an intense electric field. It has been shown that under optimal conditions the number of transformants can reach 80% of surviving cells. It is not used in humans today (Fig. 6).

Fig. 6

b) "Cell squeezing" - a method invented in 2013. It allows you to deliver molecules to cells by "soft squeezing" of the cell membrane. The method excludes the possibility of toxicity or mishandling of the target, since it does not depend on external materials or electric fields;

c) Sonoporation - a method of artificial transfer of foreign DNA into cells by acting on them with ultrasound, which causes the opening of pores in the cell membrane;
d) Optical transfection - a method in which a tiny hole is made in the membrane (about 1 micron in diameter) using a highly focused laser;
e) Hydrodynamic transfection is a method of delivery of genetic constructs, proteins, etc. by a controlled increase in pressure in the capillaries and intercellular fluid, which causes a short-term increase in the permeability of cell membranes and the formation of temporary pores in them. It is carried out by rapid injection into the tissue, while delivery is non-specific. Delivery efficiency for skeletal muscle - 22 to 60% ;

f) DNA microinjection - introduction into the nucleus of animal cells using thin glass microtubules (d = 0.1-0.5 microns). The disadvantage is the complexity of the method, the probability of destruction of the nucleus or DNA is high; a limited number of cells can be transformed. Not used for humans.

3. Particle-based methods.

a) A direct approach to transfection - a gene gun, while DNA is linked into a nanoparticle with inert solids (usually gold, tungsten), which then "shoots" directed into the nuclei of target cells. This method is applied in vitro and in vivo for the introduction of genes, in particular, into the cells of muscle tissue, for example, in a disease such as Duchenne muscular dystrophy. The size of gold particles is 1-3 microns (Fig. 7).

Fig. 7

b) Magnetofection - a method that uses the forces of magnetism to deliver DNA to target cells. First, nucleic acids (NK) are associated with magnetic nanoparticles, and then, under the influence of a magnetic field, the particles are driven into the cell. The efficiency is almost 100%, there is a clear non-toxicity. Within 10-15 minutes, particles are registered in the cell - this is much faster than other methods.
c) Impalefection ("impalement", lit. "impaling" + "infection") - a delivery method using nanomaterials such as carbon nanotubes and nanofibers. In this case, the cells are literally pierced by a bed of nanofibrils. The prefix "nano" is used to denote their very small size (within billionths of a meter) (Fig. 8).

Fig. 8

Separately, it is worth highlighting such a method as RNA transfection: not DNA is delivered to the cell, but RNA molecules - their "successors" in the protein biosynthesis chain; this activates special proteins that cut RNA into short fragments - the so-called. small interfering RNAs (miRNAs). These fragments bind to other proteins and, ultimately, this leads to inhibition of the expression of the corresponding genes by the cell. Thus, it is possible to block the action of those genes in the cell that potentially do more harm than good at the moment. RNA transfection has found wide application, in particular, in oncology.

The basic principles of gene delivery using plasmid vectors are discussed. Now you can move on to the consideration of viral methods. Viruses are non-cellular life forms, most often consisting of a nucleic acid molecule (DNA or RNA) wrapped in a protein shell. If we cut out all those sequences that cause the onset of diseases from the genetic material of the virus, then the entire virus can also be successfully turned into a "vehicle" for our gene.

The process of introducing DNA into a cell, mediated by a virus, is called transduction.
In practice, retroviruses, adenoviruses and adeno-associated viruses (AAV) are most commonly used. The first step is to figure out what the ideal candidate for transduction among viruses should be. The criteria are that it should be:

Stable;
... capacious, that is, contain a sufficient amount of DNA;
... inert with respect to the metabolic pathways of the cell;
... exact - ideally, it should integrate its genome into a specific locus of the host's nucleus genome, etc.

In real life, it is very difficult to combine at least a few points, so usually the choice occurs when considering each individual case separately (Fig. 9).

Fig. 9

Of the three most commonly used viruses listed, AAV is the safest and most accurate at the same time. Their almost only drawback is their relatively small capacity (approx. 4800 bp), which, however, turns out to be sufficient for many genes. .

In addition to the above methods, gene therapy is often used in combination with cell therapy: first, a culture of certain human cells is planted in a nutrient medium, after that, in one way or another, the necessary genes are introduced into the cells, they are cultured for a while and then transplanted into the host's body again. As a result, cells can be returned to their normal properties. So, for example, they modified human white blood cells (leukocytes) in case of leukemia (Fig. 10).

Fig. 10

The fate of a gene after it enters a cell

Since with viral vectors everything is more or less clear, due to their properties, it is more efficient to deliver genes to the final goal - the nucleus, we will dwell on the fate of the plasmid vector.

At this stage, we have achieved that DNA has passed the first large barrier - the cytoplasmic membrane of the cell.

Further, in combination with other substances, shell or without, it needs to reach the cell nucleus so that a special enzyme - RNA polymerase - synthesizes a messenger RNA (mRNA) molecule on a DNA matrix (this process is called transcription). Only after that, the mRNA will enter the cytoplasm, form a complex with ribosomes and, according to the genetic code, a polypeptide is synthesized - for example, a vascular growth factor (VEGF), which will begin to perform a certain therapeutic function (in this case, it will start the process of vascular branching in tissue prone to ischemia) ...

With regard to the expression of the introduced genes in the required type of cells, this problem is solved with the help of regulatory elements of transcription. The tissue in which expression occurs is often determined by the combination of a tissue-specific enhancer ("enhancement" sequence) with a specific promoter (the nucleotide sequence from which the RNA polymerase begins synthesis) that can be inducible ... It is known that gene activity can be modulated in vivo external signals, and since enhancers can work with any gene, insulators can also be introduced into vectors, which help the enhancer to work regardless of its position and can act as functional barriers between genes. Each enhancer contains a set of binding sites for activating or suppressing protein factors. With the help of promoters, you can also regulate the level of gene expression. For example, there are metallothionein or temperature-sensitive promoters; hormone driven promoters.

Gene expression depends on its position in the genome. In most cases, existing viral methods only lead to random insertion of a gene into the genome. To eliminate this dependence, when constructing vectors, the gene is provided with known nucleotide sequences that allow the gene to be expressed regardless of where it is inserted into the genome.

The simplest way to regulate transgene expression is to provide it with an indicator promoter that is sensitive to physiological signals such as glucose release or hypoxia. Such "endogenous" control systems can be useful in some situations, such as the implementation of glucose-dependent control of insulin production. More reliable and versatile are "exogenous" control systems, when gene expression is controlled pharmacologically by the introduction of a small drug molecule. Currently, 4 main control systems are known - those regulated by tetracycline (Tet), an insect steroid, ecdysone or its analogs, the antiprogestin drug mayfpristone (RU486), and chemical dimerizers such as rapamycin and its analogs. All of them involve drug-dependent recruitment of the transcription activation domain to the main promoter leading the desired gene, but differ in the mechanisms of this recruitment. .

Conclusion

A review of the data allows us to conclude that, despite the efforts of many laboratories in the world, all already known and tested in vivo and in vitro vector systems are far from perfect . If the problem of foreign DNA delivery in vitro practically solved, and its delivery to target cells of different tissues in vivo is successfully solved (mainly by creating constructs carrying receptor proteins, including antigens specific for certain tissues), then other characteristics of existing vector systems - integration stability, regulated expression, safety - still need serious improvements.

First of all, this concerns the stability of integration. So far, integration into the genome has been achieved only with the use of retroviral or adeno-associated vectors. The efficiency of stable integration can be increased by improving gene constructs such as receptor-mediated systems or by creating sufficiently stable episomal vectors (that is, DNA structures capable of long-term residence inside nuclei). Recently, special attention has been paid to the creation of vectors based on artificial mammalian chromosomes. Due to the presence of the main structural elements of ordinary chromosomes, such mini-chromosomes are retained for a long time in cells and are capable of carrying full-size (genomic) genes and their natural regulatory elements that are necessary for the gene to function properly, in the right tissue and at the right time.

Gene and cell therapy opens up brilliant prospects for the restoration of lost cells and tissues and the genetically engineered design of organs, which will undoubtedly significantly expand the arsenal of methods for biomedical research and create new opportunities for preserving and extending human life.

In addition, you can learn about the possibilities of modern medical science in the treatment of chromosomal abnormalities by reviewing the achievements of gene therapy. This direction is based on the implementation of the transfer of genetic material into the human body, provided that the gene is delivered to the so-called target cells using various methods.

Indications for appointment

Treatment of hereditary diseases is carried out only in the case of an exact diagnosis of the disease. At the same time, before the appointment of therapeutic measures, a number of analyzes are carried out in order to establish which hormones and other substances are produced in the body in excess, and which ones are in insufficient quantities to select the most effective dosage of drugs.

In the process of taking medications, they constantly monitor the patient's condition and, if necessary, make changes in the course of treatment.

As a rule, such patients should take medications for life or for a long period of time (for example, until the period of the end of the body growth process), and dietary recommendations should be followed strictly and constantly.

Contraindications

When developing a course of therapy, possible individual contraindications for use are taken into account and, if necessary, some drugs are replaced with others.

If a decision is made to transplant organs or tissues for some hereditary ailments, the risk of negative consequences after surgery must be taken into account.

Gene therapy is one of the rapidly developing areas of medicine, which involves the treatment of a person by introducing healthy genes into the body. Moreover, according to scientists, with the help of gene therapy, it is possible to add the 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 of the planet are potential candidates for gene therapy today, and this figure is steadily growing. And it is very gratifying that several thousand patients have already received treatment for incurable ailments as part of the ongoing trials.

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

Where gene therapy is used

Gene therapy was originally conceived to combat severe hereditary diseases such as Huntington's disease, cystic fibrosis (cystic fibrosis) and certain infectious diseases. However, 1990, when scientists managed to correct the 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 a therapy is at the very origins of development, its potential is surprising even in the scientific world.

So, for example, in addition to cystic fibrosis, modern scientists have achieved success in the fight against such hereditary pathologies as hemophilia, enzymopathy and immunodeficiency. Moreover, gene therapy allows you to fight some cancers, 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.

The principle of gene therapy

Doctors use genetic information as an active ingredient, or, to be more precise, the molecules that carry such information. Less commonly, RNA nucleic acids are used for this, and more often - DNA cells.

Each such cell has a so-called "xerox" - a mechanism by which it translates genetic information into proteins. A cell that has the correct gene and the copier works without interruptions is, from the point of view of gene therapy, a healthy cell. Each healthy cell has a whole library of original genes, which it uses for the correct and coordinated work of the whole organism. However, if, for any reason, an important gene is lost, it is not possible to recover this loss.

This becomes the cause of the development of serious genetic diseases, such as Duchenne muscular dystrophy (with it, the patient's muscle paralysis progresses, and in most cases he does not live up to 30 years old, dying from respiratory arrest). Or a less fatal situation. For example, the "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 these cases, gene therapy comes to the rescue, the task of which is to deliver a normal copy of a gene to a diseased cell and put it in a cell "copier". In this case, the work of the cell will improve, and maybe the functioning of the whole organism will be restored, thanks to which a person will get rid of a serious illness and be able to prolong his life.

What diseases does gene therapy treat?

How 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 the doctors have achieved to date is much more important. Of course, there are enough difficulties on this path, but today a number of local victories can be distinguished.

For example, modern scientists are developing approaches to the treatment of coronary heart disease through genes. But this is an incredibly common disease that affects many more people than congenital pathologies. Ultimately, a person who is faced with coronary artery disease ends up in a state where gene therapy can be the only salvation for him.

Moreover, today, with the help of genes, pathologies associated with damage to the central nervous system are treated. These are diseases such as amyotrophic lateral sclerosis, Alzheimer's disease or Parkinson's disease. Interestingly, viruses are used to treat these ailments, which tend to attack the nervous system. So, with the help of the herpes virus, cytokines and growth factors are delivered to the nervous system, which slow down the development of the disease. This is a vivid example of how a pathogenic virus, which usually causes disease, is processed in the laboratory, depriving the proteins that carry the disease, and is 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 leads the human body to an inability 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 the diseased part of the liver and correct the damaged gene, stopping further accumulation of cholesterol in the body. After that, the corrected gene is inserted into a neutralized hepatitis virus, and with its help is 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 the body for deadly diseases. Modern scientists already know how to change genes so that they stop weakening the immune system, but begin to strengthen it to resist the virus. These genes are introduced 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 an anticancer protein. Moreover, today trials are being carried out to treat brain cancer, where a gene containing information to increase the sensitivity of malignant cells to the drugs used is injected into sick patients.

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 with the progression of the disease, bones begin to collapse. Scientists have already succeeded in introducing a gene containing information on the production of this enzyme into the body of such patients.

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 the so-called Leber's atrophy, which, in fact, is a gene mutation. To date, scientists have returned visual abilities to 80 blind people by means of a modified adenovirus that delivered a “working” gene to the tissues of the eye. By the way, a few 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.

Tellingly, the method of delivering gene information using viruses is the most optimal, since viruses themselves find their targets in the body (the herpes virus will surely find neurons, and the hepatitis virus will find the liver). However, this method of gene delivery has a significant drawback - viruses are immunogens, 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 of the body, only worsening the state of health.

There is also another method for delivering gene material. It is a circular DNA molecule or plasmid. It spirals well, becoming very compact, which allows scientists to "pack" it into a chemical polymer and incorporate it into the cell. Unlike a virus, a plasmid does not trigger 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 for a long time, while the cell is "recovering".

Thus, modern scientists have two powerful methods of delivering genes to "sick" cells, and the use of viruses seems more preferable. In any case, the doctor chooses the final decision on the choice of one method or another, based on the reaction of the patient's body.

Challenges facing gene therapy

It can be concluded 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 with the genetic structure of the human body. That is why, today there is an international ban on the use of sex cells in gene therapy, as well as pre-implantation germ cells. This is done in order to prevent unwanted gene changes and mutations in our offspring.

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 main advantage of gene therapy.
Take care of yourself!

"Your child has a genetic disease" sounds like a sentence. But very often, geneticists can significantly help a sick child, and even completely compensate for some diseases. The neurologist-geneticist of the Medical Center "Pokrovsky" PBSK Bulatnikova Maria Alekseevna talks about the modern possibilities of treatment.

How common are genetic diseases?

As molecular diagnostics spread, it was discovered that the number of genetic diseases is much higher than previously thought. Many heart diseases, developmental defects, neurological abnormalities, as it turned out, have a genetic cause. In this case, I am talking specifically about genetic diseases (not predispositions), that is, conditions caused by a mutation (breakdown) in one or more genes. According to statistics, in the United States, up to a third of neurological patients are in hospitals as a result of genetic disorders. These conclusions were drawn not only by the rapid development of molecular genetics and the possibilities of genetic analysis, but also by the emergence of new neuroimaging methods, such as MRI. With the help of MRI, it is possible to determine the lesion of which area of ​​the brain leads to a disorder that has arisen in the child, and often, if we suspect a birth injury, we find changes in the structures that could not have suffered in childbirth, then an assumption arises about the genetic nature of the disease, about the improper formation of organs ... According to the results of recent studies, the influence of even difficult childbirth with undisturbed genetics can be compensated for during the first years of life.

What does knowledge about the genetic nature of the disease give?

Knowledge of the genetic causes of the disease is far from useless - it is not a sentence, but a way to find the right way to treat and correct the disorder. Many diseases are being treated successfully today; for others, genetics can offer more effective therapies that significantly improve the quality of life of the child. Of course, there are some violations that doctors cannot conquer yet, but science does not stand still, and new methods of treatment appear every day.

In my practice, there was one very typical case. An 11-year-old child consulted a neurologist regarding cerebral palsy. Upon examination and questioning of relatives, suspicions of the genetic nature of the disease appeared, which was confirmed. Fortunately for this child, the identified disease can be treated even at this age, and with the help of a change in treatment tactics, it was possible to achieve a significant improvement in the child's condition.

Currently, the number of genetic diseases, the manifestations of which can be compensated for, is constantly growing. The most famous example is phenylketonuria. It is manifested by developmental delay, oligophrenia. With the timely appointment of a diet without phenylalanine, the child grows up completely healthy, and after 20 years, the severity of the diet can be reduced. (If you are giving birth in a maternity hospital or medical center, your child will be tested for PKU in the first days of life).

The number of such diseases has increased significantly. Leucinosis also belongs to the group of metabolic diseases. In this disease, treatment should be prescribed during the first months of life (it is very important not to be late), since poisonous products of impaired metabolism lead to faster damage to the nervous tissue than with phenylketonuria. Unfortunately, if the disease is determined at the age of three months, it is impossible to completely compensate for its manifestations, but it will be possible to improve the child's quality of life. Of course, I would like this disease to be included in the screening program.

The cause of neurological disorders is often quite heterogeneous genetic lesions, precisely because there are many of them, it is so difficult to create a screening program for the timely detection of all known diseases.

These include diseases such as Pompe's, Grover's, Felidbacher's, Rett's syndrome, etc. There are many cases of a milder course of the disease.

Understanding the genetic nature of the disease allows you to direct treatment to the cause of the disorder, and not just to compensate for them, which in many cases allows you to achieve serious success and even cure the baby.

What symptoms can indicate the genetic nature of the disease?

First of all, this is a delay in the development of a child, including intrauterine (from 50 to 70% according to some estimates), myopathies, autism, refractory epileptic seizures, any malformations of internal organs. The cause of cerebral palsy can also be genetic disorders, usually in such cases, doctors talk about an atypical course of the disease. If your doctor recommends to undergo a genetic examination, do not postpone it, in this case, time is very expensive. Fading pregnancy, habitual miscarriages, including those from relatives, may also indicate the possibility of genetic abnormalities. It is very disappointing when the disease is detected too late and cannot be corrected anymore.

If the disease is not treated, do parents need to know about it?

Knowledge of the genetic nature of the disease in a child avoids the appearance of other sick children in this family. This is probably the main reason why it is worth undergoing genetic counseling even at the stage of pregnancy planning, if one of the children has developmental defects or serious illnesses. Modern science allows for both prenatal and pre-implantation genetic diagnostics, if there is information about the disease, the risk of which is present. At this stage, it is not immediately possible to check for all possible genetic diseases. Even healthy families, in which both parents have not heard of any diseases, are not immune from the appearance of children with genetic abnormalities. Recessive genes can be passed on through tens of generations and it is in your couple that they meet their half (see figure).

Should you always turn to genetics?

Genetic testing should be done based on the presence of a problem, if you or your doctor suspect. There is no need to examine a healthy child just in case. Many say that they went through all the screenings during pregnancy and everything was in order, but here ... In this case, you need to understand that screening examinations are aimed at identifying (and very effective) the most common genetic diseases - Down, Patau and Edwards diseases, mutations in individual genes, which were discussed above, with such an examination are not determined.

What is the advantage of your center?

Each genetic center has its own specialization, rather the specialization of the doctors working in it. For example, I am a pediatric neurologist by my first education. We also have a geneticist specializing in pregnancy problems. The advantage of a paid center is the doctor's ability to devote more time to his patient (an appointment lasts two hours, and the search for a solution to the problem usually continues after). There is no need to be afraid of a geneticist, this is just a specialist who can make a diagnosis that allows you to cure a seemingly hopeless disease.

"Journal of Health for Parents-to-be", No. 3 (7), 2014

Genetics in Israel is developing rapidly, there are progressive methods for the diagnosis and treatment of hereditary diseases. The range of specialized studies is constantly expanding, the laboratory base is increasing and the medical staff is improving their qualifications. The ability to diagnose and begin complex treatment of hereditary abnormalities as early as possible makes the treatment of children in Israel the most demanded and effective.

Diagnosis of genetic diseases

Treatment of hereditary diseases can be radical and palliative, but an accurate diagnosis must first be made. Thanks to the use of the latest techniques, the specialists of the Tel Aviv Sourasky Medical Center (Ichilov clinic) successfully diagnose, make an accurate diagnosis and give comprehensive recommendations for the further treatment plan.

It should be understood that if radical intervention is impossible, the efforts of doctors are aimed at improving the quality of life of a small patient: social adaptation, restoration of vital functions, correction of external defects, etc. Relief of symptoms, mapping of further actions and prediction of subsequent changes in health are all possible after making an accurate diagnosis. You can quickly undergo an examination and confirm the presence of a genetic abnormality in the Ichilov clinic, after which the patient will be prescribed a comprehensive treatment for the identified disease.

The Sourasky Center offers testing and examination not only for children, but also for future parents and pregnant women. Such a study is especially indicated for persons with a complicated personal or family history. The study will show the degree of probability of the birth of healthy offspring, after which the doctor will determine further therapeutic measures. The danger of transmitting hereditary abnormalities to a child is established as accurately as possible, using the latest technologies.

Children with genetic pathology and couples who are expecting a baby with hereditary abnormalities are given complex treatment already at the stage of collecting anamnesis and making a diagnosis.

Pediatric genetic diagnostics at Ichilov

Up to 6% of newborns have hereditary developmental abnormalities; in some children, signs of genetic disorders are detected later. Sometimes it is enough for parents to know about the existing danger in order to avoid situations that are dangerous for the child. Genetic consultations from leading Israeli specialists help to determine the presence of abnormalities at an early stage and begin treatment in a timely manner.

This includes the following diseases in children:

• malformation or multiple malformations and abnormalities (neural tube defects, cleft lip, heart defects);
• mental retardation such as autism, other developmental abnormalities of unknown etymology, child's lack of sensitivity to learning;
• structural congenital brain abnormalities;
• sensory and metabolic abnormalities;
• genetic abnormalities, diagnosed and unknown;
• chromosomal abnormalities.

Among congenital diseases, mutations in a specific gene are distinguished, which are passed from generation to generation. These include thalassemia, cystic fibrosis, and some forms of myopathies. In other cases, hereditary abnormalities are due to a change in the number or structure of chromosomes. Such a mutation can be inherited by a child from one parent or arise spontaneously, during the stage of intrauterine development. A striking example of a chromosomal disorder is Down's disease or retinoblastoma.

For the early diagnosis of hereditary defects in children, the Ichilov medical center uses various methods of laboratory research:

• molecular, allowing at the stage of intrauterine development of the fetus to establish a deviation in the DNA;
• cytogenetic, in which chromosomes in various tissues are examined;
• biochemical, which establishes metabolic deviations in the body;
• clinical, helping to establish the causes of occurrence, to carry out treatment and prevention.

In addition to prescribing complex treatment and monitoring the course of a genetic disease, the task of doctors is to predict the occurrence of the disease in the future.

Treatment of genetic diseases in children

Treatment of children in Israel consists of a whole range of activities. First of all, laboratory tests are carried out to confirm or initially diagnose. Parents will be offered the most innovative methods of technological development to identify genetic mutations.

In total, 600 genetic abnormalities are currently known to science, therefore, a timely screening of a child will help identify the disease and begin competent treatment. Genetic testing of a newborn is one of the reasons why women prefer to give birth at the Ichilov (Sourasky) clinic.

More recently, the treatment of hereditary diseases was considered a futile matter, so genetic disease was considered a sentence. Currently, significant progress has been noticeable, science is not standing still, and Israeli geneticists offer the latest treatment regimens for such deviations in the development of a child.

Genetic diseases are very heterogeneous in characteristics, therefore, treatment is prescribed taking into account the clinical manifestations and individual parameters of the patient. In many cases, inpatient treatment is preferred. Doctors should be able to conduct the most extensive examination of a small patient, choose a drug regimen, and, if indicated, carry out an operation.

To correctly select hormonal and immune therapy, you need a comprehensive examination and careful monitoring of the patient. The timing of therapeutic appointments is also individual, depending on the condition and age of the child. In some cases, parents receive a detailed plan for further procedures and monitoring of the patient. The child is selected medications to alleviate the manifestations of the disease, diet and physiotherapy.

The main directions of the treatment process in the Sourasky Center

Treatment of genetic abnormalities in children is a complex and lengthy process. It is sometimes impossible to completely cure such ailments, but the treatment is carried out in three main directions.

• The etiological method is the most effective, aimed at the causes of health disorders. The newest method of gene correction is to isolate a damaged piece of DNA, clone it and insert a healthy component into its original place. This is the most promising and innovative method of dealing with hereditary health problems. Today, the task is considered extremely difficult, but it is already being used for a number of indications.
• The pathogenetic method affects the internal processes in the body. In this case, there is an effect on the pathological genome, correction by all available methods of the physiological and biochemical state of the patient.
• The symptomatic method of action is aimed at relieving pain, negative conditions and creating obstacles for the further development of the disease. This direction is used alone or in combination with other types of treatment, but in case of identified gene disorders, it is always prescribed. Pharmacology offers a wide range of medicinal products to alleviate the manifestations of diseases. These are anticonvulsants, pain relievers, sedatives and other drugs that should be given to a child only after a medical appointment.
• A surgical method is sometimes necessary to correct external defects and internal anomalies of the child's body. Indications for surgery are prescribed very carefully. Sometimes it takes a long preliminary examination and treatment to prepare the little patient for the operation.

As a positive example of the treatment of children in Israel, one can cite statistics on a common genetic disease - autism. In the Ichilov-Sourasky hospital, early detection of anomalies (from six months of age) made it possible for 47% of these children to develop normally in the future. The revealed violations in the rest of the examined children were considered by the doctors to be insignificant and did not require medical intervention.

Parents are advised not to panic when alarming symptoms or obvious deviations in the health of their children appear. Try to contact the clinic as soon as possible, get recommendations and comprehensive advice on further actions.

Home " Postpartum period " Treatment of genetic diseases. Gene therapy: how genetic diseases are treated Can genetic diseases be cured

Human gene therapy in a broad sense involves the introduction of a functionally active gene (s) into cells in order to correct a genetic defect. There are two possible treatments for hereditary diseases. In the first case, somatic cells (cells other than reproductive cells) undergo genetic transformation. In this case, the correction of a genetic defect is limited to a specific organ or tissue. In the second case, the genotype of the germ line cells (sperm or oocytes) or fertilized oocytes (zygotes) is changed so that all the cells of the individual developing from them have "corrected" genes. As a result of gene therapy using germ line cells, genetic changes are passed down 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 established to assess the ethical and social dimensions of this issue. These were very important initiatives, since in the United States, the implementation of programs that affect the public interest is often carried out on the basis of the recommendations of such commissions. In the final opinions of both commissions, a clear line was drawn between gene therapy for somatic cells and gene therapy for germ-line cells. Somatic cell gene therapy has been classified as a standard medical intervention, similar to organ transplantation. In contrast, germ-line gene therapy was considered technologically very complex and ethically challenging to begin its practical application without delay. It was concluded that it is necessary to develop clear rules governing research in the field of gene therapy of somatic cells; the development of such documents for germline gene therapy was considered premature. In order to stop all illegal activities, it was decided to stop all experiments in the field of gene therapy for germ-line cells.

By 1985, a document was drawn up entitled "Regulations for the Drafting and Submission of Applications for Experiments in the Field of Gene Therapy of Somatic Cells." It contained all the information about what data should be presented in an application for approval of trials in the field of gene therapy of somatic cells in humans. It was based on the rules governing laboratory research with recombinant DNA; they have only been adapted for biomedical purposes.

Biomedical legislation was revised and supplemented in the 1970s. in response to the publication in 1972 of the results of a 40-year experiment by the US National Health Service in Alabama on a group of 400 illiterate African American patients with syphilis. The experiment was set up in order to study the natural development of the specified sexually transmitted disease, no treatment was carried out. The news of such a monstrous experience on uninformed people shocked many in the United States. Congress immediately ended the experiment and passed a law forbidding such research ever 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 likelihood of treatment success?
• 6. How will patients be selected for clinical trials?
• 7. Will this selection be impartial and representative?
• 8. How will patients be informed about the trial?
• 9. What kind of information should be given to them?
• 10. How will their consent be obtained?
• 11. How will the confidentiality of patient information and research be guaranteed?

When gene therapy experiments were just beginning, most clinical trial applications were first reviewed by the Ethics Committee of the institution where the research was to be carried out, and only then were they sent to the Human Gene Therapy Subcommittee. The latter assessed the applications in terms of their scientific and medical significance, compliance with the current rules, and the credibility of the arguments. If the application was rejected, it was returned back with the necessary comments. Proposers could revise the proposal and revise it. If approved, the Subcommittee on Gene Therapy discussed it in public comments 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 quality control of its purity, as well as to what kind of preclinical tests were carried out 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 initial approval process was deemed unnecessarily time-consuming and redundant. Consequently, after 1997, the Gene Therapy Subcommittee was no longer one of the agencies overseeing research in the field of human gene therapy. If the Subcommittee does exist, it will most likely become the organizer of forums to discuss ethical issues related to human gene therapy. In the meantime, the requirement that all applications in the field of gene therapy must be discussed publicly has been removed. The agency responsible for controlling the production and use of biological products conducts all necessary assessments confidentially to ensure that developers' ownership rights are respected. Currently, human gene therapy is considered a safe medical procedure, although not particularly effective. Previous concerns have dissipated, and it has become one of the main new approaches to the treatment of human diseases.

Most experts consider the approval procedure for human somatic cell gene therapy trials in the United States to be adequate; it guarantees an impartial selection of patients and their awareness, as well as the implementation of all manipulations properly, without causing harm, both to specific patients and to the human population as a whole. Rules for the conduct of gene therapy trials are also being developed in other countries. In the United States, this was done as a result of careful weighing of each proposal. As one of the participants in a hearing organized by the Subcommittees on Gene Therapy in January 1989, Dr. Walter, said: "I do not know of any other biomedical science or technology that has been tested as extensively 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 of 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 facilitate the transfer of mutant genes to the next generation from those people who were previously unable to produce offspring or could not survive to puberty. However, this hypothesis turned out to be wrong. According to population genetics, it takes thousands of years to significantly increase the frequency of a harmful or lethal gene as a result of effective treatment. So, if a rare genetic disease occurs in one out of 100,000 viable newborns, it will take about 2,000 years after the start of effective gene therapy before the frequency of this disease doubles to 1 in 50,000.

In addition to the fact that the frequency of the lethal gene hardly increases from generation to generation, as a result of long-term treatment of all who need it, the genotype of individual individuals also remains unchanged. This point can be illustrated by an example from the history of evolution. Primates, including humans, are unable to synthesize the vital 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 a genetic defect that causes the inability to biosynthesize vitamin C has not "hindered" the successful evolution of primates over millions of years. Likewise, correcting other genetic defects will not lead to a significant accumulation of "unhealthy" genes in future generations.

Gene therapy for germ line cells.

Experiments in the field of gene therapy for human germ-line cells are now strictly prohibited, but it must be admitted that some genetic diseases can only be cured in this way. The methodology of gene therapy for human germ-line cells has not yet been developed enough. However, there is no doubt that with the development of methods of genetic manipulation in animals and diagnostic testing of preimplantation embryos, this gap will be filled. Moreover, as gene therapy for somatic cells becomes an increasingly routine procedure, this will affect people's attitudes towards gene therapy for human germ-line cells, and after a while, it will become necessary to test it. We can only hope that by that time all the problems associated with the consequences of the practical application of gene therapy for human germline cells, including social and biological ones, will be resolved.

It is believed that human gene therapy can help treat serious medical conditions. Indeed, it is capable of providing 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 emerging medical field, gene therapy for human germline cells raises many questions, namely:

• 1. What is the cost of developing and implementing methods of gene therapy for human germline cells?
• 2. Should the government set priorities for medical research?
• 3. Will the priority development of gene therapy for germ-line cells lead to the curtailment of the search for 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 appropriate 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 obtain 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 cloning a frog to humans. Many articles on this topic appeared in the press, science fiction works were even written. One of the stories was about the cloning of the treacherously assassinated US President John F. Kennedy, but the more popular topic was the cloning of villains. Works about human cloning were not only implausible, but also promoted the erroneous and very dangerous idea that personality, character and other qualities of a person are due solely to his genotype. In fact, a person as a person is formed under the influence of both his genes and environmental conditions, in particular cultural traditions. For example, the vicious racism that Hitler preached is an acquired behavioral quality that is not determined by any one gene or their combination. In a different environment with different cultural characteristics, a "cloned Hitler" would not necessarily have formed a person like 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.

With the development of methods of reproductive biology in mammals and the creation of various transgenic animals, it became more and more obvious that human cloning is a matter of the not so distant future. The speculation became reality in 1997 when a sheep named Dolly was cloned. For this, the nucleus of a differentiated cell of a donor pregnant 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 does not work for other mammals, it does not seem to take too much experimentation to develop a suitable method. As a result, human cloning will immediately become the subject of any discussion that touches on the 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 it may not be all that necessary. Cloning can have positive medical and social benefits, justifying its implementation in exceptional cases. For example, it can be vital for the parents of a sick child. Liability for human cloning experiments in many countries is regulated by law, and all research related to human cloning is prohibited. Such restrictions are enough to exclude the possibility of cloning people. However, the question of the inevitability of human cloning will certainly arise.

Note!

This work was presented for the competition of popular science articles in the category "Best Review".

Deadly claws

Humanity faced this mysterious disease even before our era. Scientists in various parts of the world tried to understand and treat her: in Ancient Egypt - Ebers, in India - Sushruta, Greece - Hippocrates. All of them and many other doctors were fighting a dangerous and serious enemy - cancer. And although this battle continues to this day, it is difficult to determine if there is a chance of complete and final victory. After all, the more we study the disease, the more often questions arise - is it possible to completely cure cancer? How to avoid illness? Can treatment be made fast, affordable and inexpensive?

Thanks to Hippocrates and his observation (it was he who saw the similarity between the tumor and the tentacles of cancer), the term appeared in ancient medical treatises carcinoma(Greek carcinos) or crayfish(Latin cancer). In medical practice, malignant neoplasms are classified in different ways: carcinomas (from epithelial tissues), sarcomas (from connective, muscle tissues), leukemia (in the blood and bone marrow), lymphomas (in the lymphatic system) and others (develop in other types of cells, for example, glioma - brain cancer). But in everyday life the term "cancer" is more popular, which means any malignant tumor.

Mutations: Die or Live Forever?

Numerous genetic studies have revealed that the emergence of cancer cells is the result of genetic changes. Errors in DNA replication (copying) and repair (error correction) lead to changes in genes, including those that control cell division. The main factors that contribute to damage to the genome, and in the future to the acquisition of mutations, are endogenous (attack by free radicals formed during metabolism, chemical instability of some DNA bases) and exogenous (ionizing and UV radiation, chemical carcinogens). When mutations are fixed in the genome, they promote the transformation of normal cells into cancerous ones. Such mutations mainly occur in protooncogenes, which normally stimulate cell division. As a result, a permanently "switched on" gene can be obtained, and mitosis (division) does not stop, which, in fact, means a malignant transformation. If inactivating mutations occur in genes that normally inhibit proliferation (tumor suppressor genes), control over division is lost and the cell becomes "immortal" (Fig. 1).

Figure 1. Genetic model of cancer: colon cancer. The first step is the loss or inactivation of two alleles of the APS gene on the fifth chromosome. In the case of familiar adenomatous polyposis (FAP), one APC gene mutation is inherited. The loss of both alleles leads to the formation of benign adenomas. Subsequent gene mutations on chromosomes 12, 17, 18 of a benign adenoma can lead to transformation into a malignant tumor. A source: .

Obviously, the development of certain cancers involves changes in most or even all of these genes and can proceed in different ways. It follows from this that each tumor should be considered as a biologically unique object. Today, there are special genetic information databases on cancer, containing data on 1.2 million mutations from 8207 tissue samples belonging to 20 types of tumors: the Cancer Genome Atlas and the Catalog of Somatic Mutations in Cancer (COSMIC)).

The result of the failure of genes is uncontrolled cell division, and at subsequent stages - metastasis to various organs and parts of the body through the blood and lymphatic vessels. This is a rather complex and active process, which consists of several stages. Individual cancer cells are separated from the primary focus and are carried through the blood through the body. Then, using special receptors, they attach to endothelial cells and express proteinases that break down matrix proteins and form pores in the basement membrane. After destroying the extracellular matrix, cancer cells migrate deep into healthy tissue. Due to autocrine stimulation, they divide, forming a node (1–2 mm in diameter). With a lack of nutrition, some of the cells in the node die, and such "dormant" micrometastases can remain latent in the tissues of the organ for a long time. Under favorable conditions, the node grows, the vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGFb) gene are activated in the cells, and angiogenesis (formation of blood vessels) is initiated (Fig. 2).

However, cells are armed with special mechanisms that protect against the development of tumors:

Traditional methods and their disadvantages

If the body's defense systems failed, and the tumor nevertheless began to develop, only the intervention of doctors can save. For a long period of time, doctors have used three main "classical" therapies:

• surgical (complete removal of the tumor). It is used when the tumor is small and well localized. Some of the tissues that come into contact with the malignant neoplasm are also removed. The method is not used in the presence of metastases;
• radiation - irradiation of the tumor with radioactive particles to stop and prevent the division of cancer cells. Healthy cells are also sensitive to this radiation and often die;
• chemotherapy - drugs are used that inhibit the growth of rapidly dividing cells. Medicines also have a negative effect on normal cells.

The above approaches may not always save the patient from cancer. Often, during surgical treatment, single cancer cells remain, and the tumor can relapse, and with chemotherapy and radiation therapy, side effects occur (decreased immunity, anemia, hair loss, etc.), which lead to serious consequences, and often to the death of the patient. However, every year traditional therapies improve and new treatments appear that can defeat cancer, such as biological therapy, hormone therapy, stem cell use, bone marrow transplantation, and various supportive therapies. Gene therapy is considered the most promising, since it is aimed at the root cause of cancer - compensation for the malfunctioning of certain genes.

Gene therapy as a perspective

According to PubMed, interest in gene therapy (GT) for cancer is growing rapidly, and today GT combines a number of techniques that operate on cancer cells and in the body ( in vivo) and outside ( ex vivo) (fig. 3).

Figure 3. Two main strategies for gene therapy. Ex vivo- using vectors, genetic material is transferred into cells grown in culture (transduction), and then transgenic cells are injected into the recipient; in vivo- introduction of a vector with the desired gene into a specific tissue or organ. Picture from.

Gene therapy in vivo involves gene transfer - the introduction of genetic constructs into cancer cells or tissues that surround a tumor. Gene therapy ex vivo It consists of isolating cancer cells from a patient, inserting a therapeutic "healthy" gene into the cancer genome, and introducing the transduced cells back into the patient's body. For such purposes, special vectors created by genetic engineering methods are used. As a rule, these are viruses that detect and destroy cancer cells, while remaining harmless to healthy tissues of the body, or non-viral vectors.

Viral vectors

Retroviruses, adenoviruses, adeno-associated viruses, lentiviruses, herpes viruses and others are used as viral vectors. These viruses differ in the efficiency of transduction, in their interaction with cells (recognition and infection) and DNA. The main criterion is safety and the absence of the risk of uncontrolled spread of viral DNA: if genes are inserted in the wrong place in the human genome, they can create harmful mutations and initiate tumor development. It is also important to take into account the level of expression of the transferred genes in order to prevent inflammatory or immune reactions of the body during the hypersynthesis of target proteins (Table 1).

Table 1. Viral vectors.

Vector	Short description
Measles virus	contains a negative RNA sequence that does not elicit a protective response in cancer cells
Herpes simplex virus (HSV-1)	can carry long sequences of transgenes
Lentivirus	derived from HIV, can integrate genes into non-dividing cells
Retrovirus (RCR)	incapable of independent replication, ensures efficient incorporation of foreign DNA into the genome and the persistence of genetic changes
Monkey Foamy Virus (SFV)	a new RNA vector that transfers the transgene to the tumor and stimulates its expression
Recombinant adenovirus (rAdv)	provides efficient transfection, but a strong immune response is possible
Recombinant adeno-associated virus (rAAV)	capable of transfection of many types of cells

Non-viral vectors

Non-viral vectors are also used to transfer transgenic DNA. Polymeric drug carriers - nanoparticle constructs - are used to deliver drugs with low molecular weight, such as oligonucleotides, peptides, siRNA. Due to their small size, nanoparticles are absorbed by cells and can penetrate into capillaries, which is very convenient for delivering "therapeutic" molecules to the most inaccessible places in the body. This technique is often used to inhibit tumor angiogenesis. But there is a risk of accumulation of particles in other organs, such as the bone marrow, which can lead to unpredictable consequences. The most popular non-viral DNA delivery methods are liposomes and electroporation.

Synthetic cationic liposomes are now recognized as a promising method for the delivery of functional genes. The positive charge on the particle surface allows fusion with negatively charged cell membranes. Cationic liposomes neutralize the negative charge of the DNA chain, make its spatial structure more compact and promote efficient condensation. The plasmid-liposome complex has a number of important advantages: it can accommodate genetic constructs of practically unlimited size, there is no risk of replication or recombination, and practically does not cause an immune response in the host's body. The disadvantage of this system is the short duration of the therapeutic effect, and with repeated administration, side effects may appear.

Electroporation is a popular non-viral DNA delivery method that is fairly simple and does not induce an immune response. With the help of induced electrical impulses, pores are formed on the cell surface, and plasmid DNA can easily penetrate into the intracellular space. Gene therapy in vivo using electroporation has proven effective in a number of experiments on murine tumors. In this case, any genes can be transferred, for example, genes for cytokines (IL-12) and cytotoxic genes (TRAIL), which contributes to the development of a wide range of therapeutic strategies. In addition, this approach can be effective for the treatment of both metastatic and primary tumors.

Choice of technique

Depending on the type of tumor and its progression, the most effective treatment method is selected for the patient. To date, new promising techniques for gene therapy against cancer have been developed, including oncolytic viral HT, prodrug therapy, immunotherapy, and HT using stem cells.

Oncolytic viral gene therapy

For this technique, viruses are used that, with the help of special genetic manipulations, become oncolytic - they stop multiplying in healthy cells and only affect tumor cells. A good example of such therapy is ONYX-015, a modified adenovirus that does not express the E1B protein. In the absence of this protein, the virus cannot replicate in cells with a normal p53 gene. Two vectors designed on the basis of the herpes simplex virus (HSV-1) - G207 and NV1020 - also carry mutations in several genes in order to replicate only in cancer cells. The great advantage of the technique is that during intravenous injections, oncolytic viruses are carried through the blood throughout the body and can fight metastases. The main problems that arise when working with viruses are the possible risk of an immune response in the recipient's body, as well as the uncontrolled insertion of genetic structures into the genome of healthy cells, and, as a consequence, the occurrence of a cancerous tumor.

Gene-mediated enzymatic prodrug therapy

It is based on the introduction of "suicidal" genes into the tumor tissue, as a result of which cancer cells die. These transgenes encode enzymes that activate intracellular cytostatics, TNF receptors, and other important components for the activation of apoptosis. A suicidal combination of prodrug genes should ideally meet the following requirements: controlled gene expression; correct conversion of the selected prodrug into an active anticancer agent; complete activation of the prodrug without additional endogenous enzymes.

The disadvantage of therapy is that tumors have all the protective mechanisms inherent in healthy cells, and they gradually adapt to damaging factors and the prodrug. The adaptation process is facilitated by the expression of cytokines (autocrine regulation), cell cycle regulation factors (selection of the most resistant cancer clones), the MDR gene (responsible for susceptibility to certain medications).

Immunotherapy

Thanks to gene therapy, immunotherapy has recently begun to actively develop - a new approach to treating cancer with the help of anticancer vaccines. The main strategy of the method is active immunization of the body against cancer antigens (TAA) using gene transfer technology [? 18].

The main difference between recombinant vaccines and other drugs is that they help the patient's immune system to recognize cancer cells and destroy them. In the first stage, cancer cells are obtained from the recipient's body (autologous cells) or from special cell lines (allogeneic cells), and then they are grown in a test tube. In order for these cells to be recognized by the immune system, one or more genes are introduced that produce immunostimulating molecules (cytokines) or proteins with an increased amount of antigens. After these modifications, the cells are continued to culture, then lysis is carried out and a finished vaccine is obtained.

The wide variety of viral and non-viral vectors for transgenes allows experimentation with different types of immune cells (eg, cytotoxic T cells and dendritic cells) to inhibit the immune response and regress cancer cells. In the 1990s, it was suggested that tumor infiltrating lymphocytes (TIL) are the source of cytotoxic T lymphocytes (CTL) and natural killer cells (NK) for cancer cells. Since TIL can be easily manipulated ex vivo, they became the first genetically modified immune cells to be used for cancer immunotherapy. In T cells taken from the blood of a cancer patient, genes that are responsible for the expression of receptors for cancer antigens are altered. You can also add genes for greater survival and efficient penetration of modified T cells into the tumor. With the help of such manipulations, highly active "killers" of cancer cells are created.

When it was shown that most cancers have specific antigens and are able to induce their defense mechanisms, it was hypothesized that blocking the immune system of cancer cells would facilitate tumor rejection. Therefore, for the production of most antitumor vaccines, the patient's tumor cells or special allogeneic cells are used as a source of antigens. The main problems of tumor immunotherapy are the likelihood of autoimmune reactions in the patient's body, the absence of an antitumor response, immunostimulation of tumor growth, and others.

Stem cells

A powerful tool for gene therapy is the use of stem cells as vectors for the transfer of therapeutic agents - immunostimulating cytokines, “suicidal” genes, nanoparticles, and antiangiogenic proteins. Stem cells (SC), in addition to the ability to self-renew and differentiate, have a huge advantage over other transport systems (nanopolymers, viruses): the prodrug is activated directly in tumor tissues, which avoids systemic toxicity (the expression of transgenes contributes to the destruction of only cancer cells) ... An additional positive quality is the "privileged" state of autologous SC - the used own cells guarantee 100% compatibility and increase the safety level of the procedure. But still, the effectiveness of therapy depends on the correct ex vivo transfer of the modified gene to the SC and subsequent transfer of the transduced cells into the patient's body. In addition, before applying therapy on a large scale, it is necessary to study in detail all possible ways of SC transformation into cancer cells and develop safety measures to prevent carcinogenic SC transformation.

Conclusion

Summing up, we can say with confidence that the era of personalized medicine is coming, when a certain effective therapy will be selected for the treatment of each cancer patient. Individual treatment programs are already being developed that provide timely and appropriate care and lead to significant improvement in the condition of patients. Evolutionary approaches for personalized oncology such as genomic analysis, targeted drug manufacturing, gene therapy for cancer, and molecular diagnostics using biomarkers are already bearing fruit.

Gene therapy is a particularly promising treatment for cancer. At the moment, clinical trials are actively underway, which often confirm the effectiveness of HT in cases where standard anti-cancer treatments - surgery, radiation therapy and chemotherapy - do not help. The development of innovative methods of HT (immunotherapy, oncolytic virotherapy, "suicide" therapy, etc.) will be able to solve the problem of high mortality from cancer, and, perhaps, in the future, the diagnosis of "cancer" will not sound like a sentence.

Cancer: find out, prevent and eliminate the disease.

Literature

1. Williams S. Klag, Michael R. Cummingm. The world of biology and medicine. Fundamentals of Genetics. Moscow: Technosphere, 2007. - 726 p;
2. Bioinformatics: big databases versus big P;
3. Cui H., Cruz-Correa M. et al. (2003).

Gene therapy is one of the rapidly developing areas of medicine, which involves the treatment of a person by introducing healthy genes into the body. Moreover, according to scientists, with the help of gene therapy, it is possible to add the 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 of the planet are potential candidates for gene therapy today, and this figure is steadily growing. And it is very gratifying that several thousand patients have already received treatment for incurable ailments as part of the ongoing trials.

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

Where gene therapy is used

Gene therapy was originally conceived to combat severe hereditary diseases such as Huntington's disease, cystic fibrosis (cystic fibrosis) and certain infectious diseases. However, 1990, when scientists managed to correct the 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 a therapy is at the very origins of development, its potential is surprising even in the scientific world.

So, for example, in addition to cystic fibrosis, modern scientists have achieved success in the fight against such hereditary pathologies as hemophilia, enzymopathy and immunodeficiency. Moreover, gene therapy allows you to fight some cancers, 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.

The principle of gene therapy

Doctors use genetic information as an active ingredient, or, to be more precise, the molecules that carry such information. Less commonly, RNA nucleic acids are used for this, and more often - DNA cells.

Each such cell has a so-called "xerox" - a mechanism by which it translates genetic information into proteins. A cell that has the correct gene and the copier works without interruptions is, from the point of view of gene therapy, a healthy cell. Each healthy cell has a whole library of original genes, which it uses for the correct and coordinated work of the whole organism. However, if, for any reason, an important gene is lost, it is not possible to recover this loss.

This becomes the cause of the development of serious genetic diseases, such as Duchenne muscular dystrophy (with it, the patient's muscle paralysis progresses, and in most cases he does not live up to 30 years old, dying from respiratory arrest). Or a less fatal situation. For example, the "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 these cases, gene therapy comes to the rescue, the task of which is to deliver a normal copy of a gene to a diseased cell and put it in a cell "copier". In this case, the work of the cell will improve, and maybe the functioning of the whole organism will be restored, thanks to which a person will get rid of a serious illness and be able to prolong his life.

What diseases does gene therapy treat?

How 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 the doctors have achieved to date is much more important. Of course, there are enough difficulties on this path, but today a number of local victories can be distinguished.

For example, modern scientists are developing approaches to the treatment of coronary heart disease through genes. But this is an incredibly common disease that affects many more people than congenital pathologies. Ultimately, a person who is faced with coronary artery disease ends up in a state where gene therapy can be the only salvation for him.

Moreover, today, with the help of genes, pathologies associated with damage to the central nervous system are treated. These are diseases such as amyotrophic lateral sclerosis, Alzheimer's disease or Parkinson's disease. Interestingly, viruses are used to treat these ailments, which tend to attack the nervous system. So, with the help of the herpes virus, cytokines and growth factors are delivered to the nervous system, which slow down the development of the disease. This is a vivid example of how a pathogenic virus, which usually causes disease, is processed in the laboratory, depriving the proteins that carry the disease, and is 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 leads the human body to an inability 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 the diseased part of the liver and correct the damaged gene, stopping further accumulation of cholesterol in the body. After that, the corrected gene is inserted into a neutralized hepatitis virus, and with its help is 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 the body for deadly diseases. Modern scientists already know how to change genes so that they stop weakening the immune system, but begin to strengthen it to resist the virus. These genes are introduced 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 an anticancer protein. Moreover, today trials are being carried out to treat brain cancer, where a gene containing information to increase the sensitivity of malignant cells to the drugs used is injected into sick patients.

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 with the progression of the disease, bones begin to collapse. Scientists have already succeeded in introducing a gene containing information on the production of this enzyme into the body of such patients.

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 the so-called Leber's atrophy, which, in fact, is a gene mutation. To date, scientists have returned visual abilities to 80 blind people by means of a modified adenovirus that delivered a “working” gene to the tissues of the eye. By the way, a few 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.

Tellingly, the method of delivering gene information using viruses is the most optimal, since viruses themselves find their targets in the body (the herpes virus will surely find neurons, and the hepatitis virus will find the liver). However, this method of gene delivery has a significant drawback - viruses are immunogens, 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 of the body, only worsening the state of health.

There is also another method for delivering gene material. It is a circular DNA molecule or plasmid. It spirals well, becoming very compact, which allows scientists to "pack" it into a chemical polymer and incorporate it into the cell. Unlike a virus, a plasmid does not trigger 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 for a long time, while the cell is "recovering".

Thus, modern scientists have two powerful methods of delivering genes to "sick" cells, and the use of viruses seems more preferable. In any case, the doctor chooses the final decision on the choice of one method or another, based on the reaction of the patient's body.

Challenges facing gene therapy

It can be concluded 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 with the genetic structure of the human body. That is why, today there is an international ban on the use of sex cells in gene therapy, as well as pre-implantation germ cells. This is done in order to prevent unwanted gene changes and mutations in our offspring.

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 main advantage of gene therapy.
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