Treatment of genetic diseases. Gene therapy: how to treat genetic diseases. Is it possible to cure genetic diseases?

Human gene therapy, in a broad sense, involves introducing a functionally active gene(s) into cells to correct a genetic defect. There are two possible ways to treat hereditary diseases. In the first case, somatic cells (cells other than germ cells) are subjected to genetic transformation. In this case, correction of a genetic defect is limited to a specific organ or tissue. In the second case, the genotype of germline cells (sperm or eggs) or fertilized eggs (zygotes) is changed so that all the cells of the individual that develops from them have the “corrected” genes. Through gene therapy using germline cells, genetic changes are passed on from generation to generation.

Gene Therapy Policy somatic cells.

In 1980, representatives of the Catholic, Protestant and Jewish communities in the United States wrote an open letter to the President outlining their views on the use of genetic engineering in relation to humans. A Presidential Commission and a Congressional Commission were created to evaluate the ethical and social aspects of this problem. They were very important initiatives, since in the United States the enactment of programs affecting the public interest is often carried out on the basis of the recommendations of such commissions. The final conclusions of both commissions drew a clear distinction between gene therapy of somatic cells and gene therapy of germline cells. Gene therapy of somatic cells has been classified as standard methods medical intervention into the body, similar to organ transplantation. In contrast, germline cell gene therapy has been considered technologically too difficult and ethically too challenging to implement immediately. It was concluded that there is a need to develop clear rules governing research in the field of gene therapy of somatic cells; the development of similar documents in relation to gene therapy of germline cells was considered premature. In order to stop all illegal activities, it was decided to stop all experiments in the field of gene therapy of germline cells.

By 1985, they had developed a document entitled “Regulations on the preparation and submission of applications for experiments in the field of gene therapy of somatic cells.” It contained all the information about what data must be submitted in an application for permission to test somatic cell gene therapy in humans. The basis was taken from the rules governing laboratory research with recombinant DNA; they have only been adapted for biomedical purposes.

Biomedical legislation was revised and expanded in the 1970s. in response to the 1972 release of the results of a 40-year experiment conducted by the National Health Service in Alabama on a group of 400 illiterate African Americans with syphilis. The experiment was carried out in order to study the natural development of this sexually transmitted disease; no treatment was carried out. The news of such a horrendous experience on uninformed people shocked many in the United States. Congress immediately stopped the experiment and passed a law prohibiting such research from ever being conducted again.

Among the questions addressed to persons who applied for permission to experiment in the field of gene therapy of somatic cells were the following:

• 1. What is the disease that is supposed to be treated?
• 2. How serious is it?
• 3. Are there alternative treatments?
• 4. How dangerous is the proposed treatment for patients?
• 5. What is the probability of treatment success?
• 6. How will patients be selected for clinical trials?
• 7. Will this selection be unbiased and representative?
• 8. How will patients be informed about the tests?
• 9. What kind of information should they be given?
• 10. How will their consent be obtained?
• 11. How will the confidentiality of information about patients and research be guaranteed?

When gene therapy experiments first began, most applications for clinical trials were first reviewed by the Ethics Committee of the institution where the research was to be carried out before being forwarded to the Human Gene Therapy Subcommittee. The latter assessed applications from the point of view of their scientific and medical significance, compliance with current rules, and the persuasiveness of the arguments. If the application was rejected, it was returned with the necessary comments. The authors of the proposal could review the proposal and rework it. If an application was approved, the Gene Therapy Subcommittee discussed it in public discussions using the same criteria. After approval of the application at this level, the director of the Subcommittee approved it and signed the authorization for clinical trials, without which they could not begin. In this last case Special attention addressed the method of obtaining the product, methods of qualitative control of its purity, as well as what preclinical tests were conducted to ensure the safety of the product.

But as the number of applications increased over time, and gene therapy became, in the words of one commentator, “the winning ticket in medicine,” the original application approval process was considered unnecessarily time-consuming and redundant. Accordingly, after 1997, the Gene Therapy Subcommittee was no longer one of the agencies overseeing human gene therapy research. If the Subcommittee exists, it will most likely provide forums to discuss ethical issues related to human gene therapy. In the meantime, the requirement that all gene therapy applications be discussed publicly has been lifted. The agency responsible for monitoring the production and use of biological products conducts all necessary assessments confidentially to ensure that the developers' proprietary rights are respected. Currently, human gene therapy is considered a safe medical procedure, although not particularly effective. Previously expressed concerns have dissipated, and it has become one of the main new approaches to the treatment of human diseases.

Most experts consider the approval process for human somatic cell gene therapy trials in the United States to be quite adequate; it guarantees the impartial selection of patients and their awareness, as well as the implementation of all manipulations properly, without causing harm to both specific patients and the human population as a whole. Other countries are also currently developing regulations for gene therapy trials. In the US this was done by carefully weighing each proposal. As Dr. Walters, one of the participants in the Gene Therapy Subcommittee hearings in January 1989, said: "I know of no other biomedical science or technology that has been subjected to such extensive scrutiny as gene therapy."

Accumulation of defective genes in future generations.

There is an opinion that the treatment of genetic diseases using gene therapy of somatic cells will inevitably lead to a deterioration in the gene pool of the human population. It is based on the idea that the frequency of a defective gene in a population will increase from generation to generation, since gene therapy will promote the transmission of mutant genes to the next generation from those people who were previously unable to produce offspring or could not survive to adulthood. However, this hypothesis turned out to be incorrect. According to population genetics, it takes thousands of years for a harmful or lethal gene to significantly increase in frequency as a result of effective treatment. Thus, if a rare genetic disease occurs in 1 in 100,000 live births, it will take approximately 2,000 years after the introduction of effective gene therapy before the incidence of the disease doubles to 1 in 50,000.

In addition to the fact that the frequency of the lethal gene hardly increases from generation to generation, as a result of long-term treatment of everyone who needs it, the genotype of individual individuals also remains unchanged. This point can be illustrated with an example from the history of evolution. Primates, including humans, are unable to synthesize vital vitamin C; they must obtain it from external sources. Thus, we can say that we are all genetically defective in the gene for this vital substance. In contrast, amphibians, reptiles, birds, and non-primate mammals synthesize vitamin C. Yet the genetic defect that causes the inability to biosynthesize vitamin C did not “prevent” the successful evolution of primates for more than millions of years. Likewise, correcting other genetic defects will not lead to a significant accumulation of “unhealthy” genes in future generations.

Gene therapy of germline cells.

Experiments in the field of gene therapy of human germline cells are now strictly prohibited, but it must be recognized that some genetic diseases can only be cured in this way. The methodology for gene therapy of human germline cells has not yet been sufficiently developed. However, there is no doubt that with the development of methods of genetic manipulation in animals and diagnostic testing of preimplantation embryos, this gap will be filled. Moreover, as somatic cell gene therapy becomes more routine, this will affect people's attitudes toward human germline gene therapy, and over time there will be a need to test it. One can only hope that by that time all the problems associated with the consequences of the practical use of gene therapy for human germline cells, including social and biological ones, will be resolved.

Human gene therapy is thought to help treat serious illnesses. Indeed, it can provide correction for a number of physical and mental disorders, although it remains unclear whether society will find such use of gene therapy acceptable. Like any new medical field, gene therapy of human germline cells raises numerous questions, including:

• 1. What is the cost of developing and implementing gene therapy methods for human germline cells?
• 2. Should the government set medical research priorities?
• 3. Will the priority development of gene therapy for germline cells lead to the curtailment of efforts to find other methods of treatment?
• 4. Will it be possible to reach all patients who need it?
• 5. Will an individual or company be able to obtain exclusive rights to treat specific diseases using gene therapy?

Human cloning.

Public interest in the possibility of human cloning arose in the 1960s, after corresponding experiments were carried out on frogs and toads. These studies showed that the nucleus of a fertilized egg can be replaced with the nucleus of an undifferentiated cell, and the embryo will develop normally. Thus, in principle, it is possible to isolate nuclei from undifferentiated cells of an organism, introduce them into fertilized eggs of the same organism, and produce offspring with the same genotype as the parent. In other words, each of the descendant organisms can be considered a genetic clone of the original donor organism. In the 1960s it seemed that, despite the lack of technical capabilities, it was not difficult to extrapolate the results of frog cloning to humans. Many articles on this topic appeared in the press, and even science fiction works were written. One of the stories was about the cloning of the treacherously assassinated US President John F. Kennedy, but a more popular topic was the cloning of villains. The works about human cloning were not only implausible, but also promoted the erroneous and very dangerous idea that a person’s personality traits, character and other qualities are determined solely by his genotype. In fact, a person as a personality is formed under the influence of both his genes and environmental conditions, in particular cultural traditions. For example, the malicious racism that Hitler preached is an acquired behavioral quality that is not determined by any one gene or their combination. In another environment with different cultural characteristics, the “cloned Hitler” would not necessarily have formed into a person similar to the real Hitler. Likewise, a “clone of Mother Teresa” would not necessarily “make” a woman who dedicated her life to helping the poor and sick in Calcutta.

As methods of mammalian reproductive biology developed and the creation of various transgenic animals, it became increasingly clear that human cloning was a matter of the not-too-distant future. The speculation became reality in 1997, when a sheep named Dolly was cloned. For this purpose, the nucleus of a differentiated cell from a donor sheep was used. The methodological approach that was used to “create” Dolly is, in principle, suitable for obtaining clones of any mammals, including humans. And even if it doesn't work out well in other mammal species, it probably won't take too much experimentation to develop a suitable method. As a result, human cloning will immediately become the subject of any discussion involving ethical problems of genetics and biological medicine.

Without a doubt, human cloning is a complex and controversial issue. For some, the very idea of ​​creating a copy of an already existing individual through experimental manipulation seems unacceptable. Others believe that a cloned individual is the same as an identical twin, despite the age difference, and therefore cloning is not inherently malicious, although perhaps not entirely necessary. Cloning can have positive medical and social effects that justify its implementation in exceptional cases. For example, it may be vital for the parents of a sick child. Liability for human cloning experiments is regulated by law in many countries, and all research related to human cloning is prohibited. Such restrictions are enough to exclude the possibility of human cloning. However, the question of the inevitability of human cloning will certainly arise.

Gene therapy in the broad sense of the word means treatment by introducing semantic DNA sequences into the patient's tissues or cells. Initially, gene therapy was seen as a way to correct a defect in a gene.

Further research corrected these ideas. It turned out that it is much easier to correct not the defect in the gene itself, but to carry out the correction by introducing a fully functioning gene into the patient’s body. It turned out that gene therapy should be carried out exclusively on somatic tissues; gene therapy at the level of germ and germ cells is very problematic and unrealistic. The reason for this is the real danger of clogging the gene pool with unwanted artificial gene constructs or introducing mutations with unpredictable consequences for the future of humanity (Fr. Anderson, T. Caskey, Fr. Collins, etc.). Finally, the practical methodology of gene therapy has proven to be suitable for treating not only monogenic hereditary diseases, but also widespread diseases such as malignant tumors, severe forms viral infections, AIDS, cardiovascular and other diseases.

The first clinical trials of gene therapy were undertaken on May 22, 1989, with the goal of genetically marking tumor-infiltrating lymphocytes in advanced melanoma. The first monogenic hereditary disease for which gene therapy methods were applied was hereditary immunodeficiency caused by a mutation in the adenosine deaminase gene. With this disease, 2-deoxyadenosine accumulates in the blood of patients in high concentrations, which has a toxic effect on T and B lymphocytes, resulting in the development of severe combined immunodeficiency. On September 14, 1990, in Bethesda (USA), a 4-year-old girl suffering from this rather rare disease (1:100,000) was transplanted with her own lymphocytes, previously transformed ex vivo with the ADA gene (ADA gene + marker gene PEO + retroviral vector). The therapeutic effect was observed for several months, after which the procedure was repeated at intervals of 3-5 months. During 3 years of therapy, a total of 23 intravenous transfusions of ADA-transformed lymphocytes were performed. As a result of treatment, the patient's condition improved significantly.

Other monogenic hereditary diseases for which there are already officially approved protocols and clinical trials have begun relate to familial hypercholesterolemia (1992), hemophilia B (1992), cystic fibrosis (1993), Gaucher disease (1993). By 1993, in the United States alone, 53 projects were approved for clinical trials of genetically engineered designs. By 1995, the number of such projects worldwide had increased to 100, and more than 400 patients were directly involved in these studies. At the same time, even today's gene therapy research takes into account that the consequences of manipulating genes or recombinant DNA in vivo have not been sufficiently studied. Therefore, when developing gene therapy programs, the safety of treatment regimens for both the patient and the population as a whole is of fundamental importance.

The gene therapy program for clinical trials includes the following sections: justification for the choice of nosology for conducting a course of gene therapy; determination of the type of cells subject to genetic modification; scheme for constructing exogenous DNA; substantiation of the biological safety of the introduced gene construct, including experiments on cell cultures and model animals; development of a procedure for transferring it into patient cells; methods for analyzing the expression of introduced genes; assessment of clinical (therapeutic) effect; possible side effects and ways to prevent them.

In Europe, such protocols are drawn up and approved in accordance with the recommendations of the European working group on gene transfer and gene therapy. The most important element in a gene therapy program is the analysis of the consequences of the procedures performed. The decisive condition for successful gene therapy is to ensure effective delivery, that is, transfection or transduction (using viral vectors) of a foreign gene into target cells, ensuring its long-term persistence in these cells and creating conditions for full operation, that is, expression. The key to long-term persistence of foreign DNA in recipient cells is its integration into the genome, that is, into the host DNA cells. The main methods of delivering foreign genes into cells are divided into chemical, physical and biological. The construction of virus-based vectors is the most interesting and promising branch of gene therapy.

The emergence of fundamentally new technologies that make it possible to actively manipulate genes and their fragments, ensuring targeted delivery of new blocks of genetic information to specified areas of the genome, has revolutionized biology and medicine. In this case, the gene itself increasingly begins to act as a medicine used to treat various diseases. The use of gene therapy to combat multifactorial diseases is not far off. Already now, at the current level of our knowledge about the human genome, such modifications by gene transfection are quite possible, which can be undertaken in order to improve a number of physical (for example, height), mental and intellectual parameters. Thus, modern human science, in its new round of development, has returned to the idea of ​​“improving the human race,” postulated by the outstanding English geneticist Fr. Galton and his students.

Gene therapy in the 21st century not only offers real ways to treat severe hereditary and non-hereditary diseases, but also, in its rapid development, poses new problems for society that need to be solved in the near future.

Note!

This work was submitted to the competition of popular science articles in the “Best Review” category.

Deadly claws

Humanity was faced with this mysterious disease even before our era. Scientists in various parts of the world tried to understand and treat it: in Ancient Egypt - Ebers, in India - Sushruta, Greece - Hippocrates. All of them and many other doctors fought against a dangerous and serious enemy - cancer. And although this battle continues to this day, it is difficult to determine whether 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? Is it possible to make treatment quick, accessible and inexpensive?

Thanks to Hippocrates and his powers of observation (it was he who saw the similarity between a tumor and the tentacles of cancer), the term appeared in ancient medical treatises carcinoma(Greek carcinos) or cancer(lat. cancer). In medical practice, malignant neoplasms are classified differently: 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 research discovered that the occurrence 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 genome damage, and subsequently to the acquisition of mutations, are endogenous (attack of free radicals formed during metabolism, chemical instability of some DNA bases) and exogenous (ionizing and UV radiation, chemical carcinogens). When mutations become established in the genome, they promote the transformation of normal cells into cancer cells. Such mutations mainly occur in proto-oncogenes, which normally stimulate cell division. As a result, the gene may be constantly “on”, and mitosis (division) does not stop, which, in fact, means malignant degeneration. 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. When familial cancer(familiar adenomatous polyposis, FAP) one mutation of the APC gene is inherited. Loss of both alleles leads to the formation of benign adenomas. Subsequent mutations of genes on chromosomes 12, 17, 18 of a benign adenoma can lead to transformation into a malignant tumor. Source: .

It is clear that the development of certain types of cancer involves changes in most or even all of these genes and can occur 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 related to 20 types of tumors: the Cancer Genome Atlas and the catalog somatic mutations in cancer (Catalog of Somatic Mutations in Cancer (COSMIC)).

The result of a malfunction of genes is uncontrolled cell division, and in subsequent stages - metastasis into various organs and parts of the body through blood and lymphatic vessels. This is a rather complex and active process that consists of several stages. Individual cancer cells are separated from the primary site and spread through the blood throughout the body. Then, using special receptors, they attach to endothelial cells and express proteinases, which break down matrix proteins and form pores in the basement membrane. Having destroyed the extracellular matrix, cancer cells migrate deeper healthy tissue. Due to autocrine stimulation, they divide to form 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 quite a long time. Under favorable conditions, the node grows, the gene for vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGFb) is activated in the cells, and angiogenesis is initiated (formation blood vessels) (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 fail and the tumor nevertheless begins to develop, only medical intervention can save it. For a long period, doctors have used three main “classical” therapies:

• surgical ( complete removal tumors). Used when the tumor is small and well localized. Part of the tissue that is in contact with the malignant tumor is 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 to inhibit the growth of rapidly dividing cells. Medicines also have a negative effect on normal cells.

The approaches described above cannot always save a patient from cancer. Often when surgical treatment single cancer cells remain, and the tumor can recur, 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 treatments are improving and new treatments are emerging that can defeat cancer, such as biological therapy, hormonal therapy, the use of stem cells, bone marrow transplantation, and various supportive therapies. Gene therapy is considered the most promising, as it is aimed at the root cause of cancer - compensation for the malfunction of certain genes.

Gene therapy as a prospect

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

Figure 3. Two main gene therapy strategies. Ex vivo- genetic material is transferred using vectors into cells grown in culture (transduction), and then the transgenic cells are introduced 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 into tissues that surround the tumor. Gene therapy ex vivo 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 identify 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 their transduction efficiency, 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 the development of a tumor. It is also important to consider the expression level of the transferred genes to prevent inflammatory or immune reactions in the body during hypersynthesis of target proteins (Table 1).

Table 1. Viral vectors.

Vector	Short description
Measles virus	contains a negative RNA sequence that does not induce 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 effective integration of foreign DNA into the genome and the persistence of genetic changes
Simian foamy virus (SFV)	a new RNA vector that transfers the transgene to the tumor and stimulates its expression
Recombinant adenovirus (rAdv)	ensures efficient transfection, but a strong immune reaction is possible
Recombinant adeno-associated virus (rAAV)	capable of transfecting many cell types

Non-viral vectors

Non-viral vectors are also used to transfer transgenic DNA. Polymer drug carriers - nanoparticle structures - are used to deliver drugs with low molecular weight, for example, oligonucleotides, peptides, siRNA. Due to their small size, nanoparticles are absorbed by cells and can penetrate capillaries, which is very convenient for delivering “medicinal” molecules to the most inaccessible places in the body. This technique is often used to inhibit tumor angiogenesis. But there is a risk of particles accumulating 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 currently recognized as a promising method for delivering functional genes. The positive charge on the surface of the particles ensures fusion with negatively charged cell membranes. Cationic liposomes neutralize the negative charge of the DNA chain, make its spatial structure more compact and promote effective condensation. The plasmid-liposome complex has a number of important advantages: it can accommodate genetic constructs of almost unlimited size, there is no risk of replication or recombination, and it practically does not cause an immune response in the host body. The disadvantage of this system is the short duration of the therapeutic effect, and side effects may appear with repeated administration.

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

Selection of equipment

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

Oncolytic viral gene therapy

This technique uses viruses that, with the help of special genetic manipulations, become oncolytic - they stop reproducing in healthy cells and affect only tumor cells. A good example 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 based on the herpes simplex virus (HSV-1) - G207 and NV1020 - also carry mutations in several genes 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 possible risk the occurrence of an immune response in the recipient’s body, as well as the uncontrolled integration of genetic constructs into the genome of healthy cells, and, as a consequence, the occurrence of a cancerous tumor.

Gene-mediated enzyme prodrug therapy

It is based on the introduction of “suicide” genes into the tumor tissue, as a result of which the cancer cells die. These transgenes encode enzymes that activate intracellular cytostatics, TNF receptors and other important components for the activation of apoptosis. A suicidal prodrug gene combination 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 contain all defense mechanisms, characteristic of healthy cells, and they gradually adapt to damaging factors and prodrugs. The adaptation process is facilitated by the expression of cytokines (autocrine regulation), cell cycle regulation factors (selection of the most resistant cancer clones), and the MDR gene (responsible for susceptibility to certain medications).

Immunotherapy

Thanks to gene therapy, immunotherapy has recently begun to actively develop - new approach for the treatment of cancer using antitumor 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 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 grown in vitro. In order for these cells to be recognized by the immune system, one or more genes are introduced that produce immune-stimulating molecules (cytokines) or proteins with an increased number of antigens. After these modifications, the cells continue to be cultured, then lysed and the finished vaccine is obtained.

The wide variety of viral and nonviral vectors for transgenes allows experimentation on different types of immune cells (eg, cytotoxic T cells and dendritic cells) to inhibit the immune response and regression of cancer cells. In the 1990s, it was proposed that tumor infiltrating lymphocytes (TILs) are a source of cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells for cancer cells. Since TIL can be easily manipulated ex vivo, they became the first genetically modified immune cells, which have been used for anticancer immunotherapy. In T cells removed from the blood of a cancer patient, genes that are responsible for the expression of receptors for cancer antigens are changed. Genes can also be added to make the modified T cells more likely to survive and enter the tumor more efficiently. With the help of such manipulations, highly active “killers” of cancer cells are created.

When it was proven that most cancers have specific antigens and are able to induce their own 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, patient 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, suicide genes, nanoparticles and antiangiogenic proteins. Stem cells (SC), in addition to the ability to self-renew and differentiate, have a huge advantage compared to other transport systems (nanopolymers, viruses): activation of the prodrug occurs directly in tumor tissues, which avoids systemic toxicity (expression of transgenes contributes to the destruction of only cancer cells) . An additional positive quality is the “privileged” state of autologous SCs - the used own cells guarantee 100% compatibility and increase the level of safety 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 transduced cells into the patient’s body. In addition, before using therapy on a large scale, it is necessary to study in detail all possible ways of transformation of SC into cancer cells and develop safety measures to prevent carcinogenic transformation of SC.

Conclusion

To summarize, we can confidently say that the era of personalized medicine is coming, when a specific effective therapy will be selected for the treatment of each cancer patient. Individual treatment programs are already being developed that provide timely and correct care and lead to significant improvement in the patient’s condition. Evolutionary approaches for personalized oncology, such as genomic analysis, targeted drug production, cancer gene therapy and molecular diagnostics using biomarkers are already bearing fruit.

A particularly promising method of treating cancer is gene therapy. On this moment Clinical trials are actively being conducted, which often confirm the effectiveness of HT in cases where standard anticancer treatment - surgery, radiation therapy and chemotherapy - does not help. Development innovative techniques GT (immunotherapy, oncolytic virotherapy, “suicidal” 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 death sentence.

Cancer: recognize, prevent and eliminate the disease.

Literature

1. Williams S. Klug, Michael R. Cummingm. World of biology and medicine. Basics of genetics. Moscow: Tekhnosphere, 2007. - 726 p.;
2. Bioinformatics: Big Databases vs. Big P;
3. Cui H., Cruz-Correa M. et al. (2003).

Introduction

Every year in scientific journals There are more and more articles about medical clinical studies in which, one way or another, 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 narrowed the lumen of blood vessels in lower limbs and as a result, persistent ischemia of the surrounding tissues has developed, that is, these tissues experience a severe lack of nutrients and oxygen, which are normally carried by blood throughout the body. It is often impossible to treat such patients with surgical manipulations and medications, but if the cells are locally forced to release more protein factors that would affect the process of formation and germination of new vessels, then ischemia would become much less pronounced and life would become much easier for patients.

Gene therapy today can be defined as the treatment of diseases by introducing genes into the cells of patients in order to specifically change gene defects or give the cells new functions. The first clinical trials of gene therapy methods were undertaken quite recently - on May 22, 1989, for the purpose of diagnosing cancer. The first hereditary disease for which gene therapy methods 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 reached 2 thousand.

At the same time, in modern research on gene therapy, it is necessary to take into account that the consequences of manipulating genes or “shuffled” (recombinant) DNA in vivo(Latin literally “in the living”) 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 relevant committees and commissions. In the USA, these are the Recombinant DNA Advisory Committee (RAC) and the Drug Administration. food products(Food and Drug Administration, FDA) with subsequent mandatory approval of the project by the director National Institutes health (National Institutes of Health).

So, we have decided that this treatment is based on the fact that if some tissues of the body are deficient in certain individual protein factors, then this can be corrected by introducing the corresponding genes encoding proteins into these tissues, and everything will become more or less wonderful. It will not be possible to introduce the proteins themselves, because our body will immediately react with a strong immune reaction, and the duration of action would be insufficient. Now you need to decide on the method of delivering the gene into cells.

Transfection cells

First, it’s 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 properties characteristic of them:

1. Origin of replication (ori)- the sequence of nucleotides from which DNA duplication begins. If the vector DNA cannot double (replicate), then the necessary healing effect will not be achieved, because it will simply be quickly broken down by intracellular nuclease enzymes, and due to the lack of matrices, much fewer protein molecules will ultimately be formed. It should be noted that these points are specific for each biological species, that is, if vector DNA is supposed to be obtained by propagating it in a bacterial culture (and not just by chemical synthesis, which is usually much more expensive), then two separate origins of replication will be required - 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 necessary in order to stitch the vector DNA (which, in essence, is a “blank”) with the desired therapeutic genes into a single molecule. Such a molecule crosslinked from two or more parts is called “recombinant”;

3. It is clear that we would like to obtain millions of copies of a recombinant DNA molecule. Again, if we are dealing with a bacterial cell culture, then this DNA needs to be isolated. The problem is that not all bacteria will ingest the molecule we need; some will not do this. To distinguish these two groups, they insert selective markers- areas of resistance to certain chemicals; Now if you add these very substances 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 process of introducing a plasmid vector into certain cells is called transfection. A plasmid is a fairly short and usually circular DNA molecule that is found in the cytoplasm of a bacterial cell. Plasmids are not associated with the bacterial chromosome, they can replicate independently of it, can be released by the bacterium into the environment or, conversely, absorbed (the absorption process is transformation). With the help of plasmids, bacteria can exchange genetic information, for example, transmitting 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, inserting a gene insert into it and introducing it into a cell. Different inserts can be inserted into the same plasmid .

Gene therapy methods

There are two main approaches, differing 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; in this case, it is expected that the introduced material will enter all cells of the recipient (and even 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-reproductive cells of an already born person and it is not transmitted to the germ cells.

Gene therapy in vivo is based on the direct introduction of cloned (multiplied) and packaged DNA sequences in a certain way into certain tissues of the patient. Particularly promising for the treatment of gene diseases in vivo is the introduction of genes using aerosol or injected vaccines. Aerosolized gene therapy is being developed, typically for the treatment pulmonary diseases(cystic fibrosis, lung cancer).

There are many steps involved in developing a gene therapy program. This includes a thorough analysis of the tissue-specific expression of the corresponding gene (i.e., synthesis on the matrix of a gene of some protein in a certain tissue), and identification of a primary biochemical defect, and a study of the structure, function and intracellular distribution of its protein product, as well as a biochemical analysis of the pathological process. All this data is taken into account when drawing up the appropriate medical protocol.

It is important that when drawing up gene correction schemes, the efficiency of transfection and the degree of correction of the primary biochemical defect under cell culture conditions are assessed ( in vitro,"in vitro") and, most importantly, in vivo on animal biological models. Only after this can the clinical trial program begin .

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’s worth mentioning right away that chemical and physical methods(e.g. electroporation + enveloping DNA with liposomes)

Direct methods

1. Chemical-based transfection can be classified into several types: using a cyclodextrin substance, polymers, liposomes or nanoparticles (with or without chemical or viral functionalization, i.e. 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 effective absorption. Disadvantage - only about 1 - 10% of the DNA reaches the nucleus. Method used in vitro for transferring DNA into human cells (Fig. 3);

Fig.3

b) The use of highly branched organic molecules - dendrimer, to bind DNA and transfer it into the cell (Fig. 4);

Fig.4

c) Very effective method To transfect DNA, it is introduced through liposomes - small, membrane-surrounded bodies that can merge with the cell cytoplasmic membrane (CPM), which is a double layer of lipids. For eukaryotic cells, transfection is more efficient 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 they introduce into cells is usually immediately captured by lysosomes and destroyed. Introduction of DNA into human cells using liposomes is the mainstay of therapy today. in vivo(Fig. 5);

Fig.5

d) Another method is the use of cationic polymers such as diethylaminoethyl dextran or polyethylenimine. Negatively charged DNA molecules bind to positively charged polycations, and this complex further enters the cell by endocytosis. DEAE-dextran changes physical properties plasma membrane and stimulates the uptake 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, containing many positively charged amino acids (Lys, Arg), in natural conditions help compactly pack a long chain of DNA into a relatively small cell nucleus.

2. Physical methods:

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

Fig.6

b) “Cell squeezing” is a method invented in 2013. It allows you to deliver molecules into cells by “gently squeezing” the cell membrane. The method eliminates the possibility of toxicity or mis-targeting as it does not rely on external materials or electrical fields;

c) Sonoporation is a method of artificially transferring foreign DNA into cells by exposing them to ultrasound, causing 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 μm in diameter) using a highly focused laser;
e) Hydrodynamic transfection - a method of delivering 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, and delivery is non-specific. Delivery efficiency for skeletal muscle - from 22 to 60% ;

f) Microinjection of DNA - introduction into the nucleus of an animal cell using thin glass microtubules (d=0.1-0.5 µm). The disadvantage is the complexity of the method, there is a high probability of destruction of the nucleus or DNA; a limited number of cells can be transformed. Not for human use.

3. Particle-based methods.

a) A direct approach to transfection is a gene gun, in which DNA is concatenated into a nanoparticle with inert solids (usually gold, tungsten), which is then “shot” directed into the nuclei of target cells. This method is applied in vitro And in vivo for introducing genes, in particular, into muscle tissue cells, 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 is a method that uses the forces of magnetism to deliver DNA to target cells. First, nucleic acids (NA) are associated with magnetic nanoparticles, and then, under the influence magnetic field, particles are driven into the cell. The effectiveness is almost 100%, obvious non-toxicity is noted. Within 10-15 minutes, particles are registered in the cell - this is much faster than other methods.
c) Impalefection; "impalement", lit. "impalement" + "infection") - a delivery method using nanomaterials such as carbon nanotubes and nanofibers. In this case, the cells are literally punctured by a layer of nanofibrils. The prefix “nano” is used to denote their very small sizes (within billionths of a meter) (Fig. 8).

Fig.8

Separately, it is worth highlighting such a method as RNA transfection: it is not DNA that is delivered into the cell, but RNA molecules - their “successors” in the protein biosynthesis chain; in this case, special proteins are activated that cut the RNA into short fragments - the so-called. small interfering RNA (siRNA). These fragments bind to other proteins and, ultimately, this leads to inhibition of the cell's expression of the corresponding genes. In this way, 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 reviewed. Now we can move on to considering viral methods. Viruses are noncellular forms life, most often representing a nucleic acid molecule (DNA or RNA) wrapped in a protein shell. If you cut out from the genetic material of the virus all those sequences that cause diseases, 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, the most commonly used are retroviruses, adenoviruses and adeno-associated viruses (AAV). First, it’s worth figuring out what the ideal candidate for transduction among viruses should be. The criteria are that it must be:

Stable;
. capacious, that is, to accommodate a sufficient amount of DNA;
. inert in relation to the metabolic pathways of the cell;
. precise - ideally, it should integrate its genome into a specific locus of the genome of the host nucleus, etc.

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

Fig.9

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

In addition to the listed 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 which the necessary genes are introduced into the cells in one way or another, cultivated for some time and again transplanted into the host’s body. As a result, the cells can be returned to their normal properties. So, for example, human white blood cells (leukocytes) were modified for leukemia (Fig. 10).

Fig.10

The fate of the gene after it enters the cell

Since everything is more or less clear with viral vectors due to their ability to more efficiently 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 big barrier - the cytoplasmic membrane of the cell.

Further, in combination with other substances, shell or not, it needs to achieve cell nucleus so that a special enzyme - RNA polymerase - synthesizes a messenger RNA (mRNA) molecule on a DNA template (this process is called transcription). Only after this the mRNA will be released into the cytoplasm, forms a complex with ribosomes and, according to the genetic code, a polypeptide is synthesized - for example, vascular growth factor (VEGF), which will begin to perform a certain therapeutic function (in this case, it will start the process of formation of vessel branching in tissue subject to ischemia) .

As for the expression of the introduced genes in the required cell type, this problem is solved with the help of transcription regulatory elements. The tissue in which expression occurs is often determined by the combination of a tissue-specific enhancer (“enhancing” sequence) with a specific promoter (a sequence of nucleotides from which RNA polymerase begins synthesis), which 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 be introduced into the 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 suppressive protein factors. Promoters can also be used to regulate the level of gene expression. For example, there are metallothionein or temperature-sensitive promoters; promoters controlled by hormones.

Expression of a gene depends on its position in the genome. In most cases, existing viral methods only result in random insertion of a gene into the genome. To eliminate such a dependence, when constructing vectors, the gene is supplied with known nucleotide sequences, which 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 a physiological signal, such as glucose release or hypoxia. Such "endogenous" control systems may be useful in some situations, such as glucose-dependent control of insulin production. “Exogenous” control systems are more reliable and universal, when gene expression is controlled pharmacologically by the introduction of a small drug molecule. Currently, 4 main control systems are known - regulated by tetracycline (Tet), the insect steroid, ecdysone or its analogs, the antiprogestin drug mayfpristone (RU486) and chemical dimerizers such as rapamycin and its analogs. All of them include drug-dependent attraction of the transcription activation domain to the main promoter leading the desired gene, but they differ in the mechanisms of this attraction .

Conclusion

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

First of all, this concerns the stability of integration. Until now, integration into the genome has only been achieved using 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 mammalian artificial chromosomes. Due to the presence of the basic structural elements of ordinary chromosomes, such mini-chromosomes are retained in cells for a long time and are able to carry full-size (genomic) genes and their natural regulatory elements, which are necessary for the correct functioning of the gene, 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 genetic engineering 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 capabilities of modern medical science in the treatment of chromosomal abnormalities by familiarizing yourself with the achievements of gene therapy. This direction is based on the transfer of genetic material into the human body, subject to the delivery of the gene to the so-called target cells using various methods.

Indications for use

Treatment of hereditary diseases is carried out only if the disease is accurately identified. At the same time, before prescribing therapeutic measures, a number of tests are carried out to determine which hormones and other substances are produced in excess in the body and which are produced in insufficient quantities in order to select the most effective dosage of drugs.

While taking medications, the patient’s condition is constantly monitored and, if necessary, changes are made to 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 end of the body’s 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, replace some drugs with others.

When deciding to transplant organs or tissues for certain hereditary diseases, 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 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 some 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 active substance doctors use genetic information, or, to be 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, the 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 essence, is 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 immune reaction 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

We can draw a certain conclusion 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 simply powerless. And this is the most important advantage of gene treatment.
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. Maria Alekseevna Bulatnikova, a neurologist-geneticist at the Pokrovsky Medical Center, PBSK, talks about modern treatment options.

How common are genetic diseases?

As molecular diagnostics have become more widespread, it has been discovered that the number of genetic diseases is much greater than previously thought. Many heart diseases, developmental defects, and neurological abnormalities appear to have a genetic cause. In this case, I am talking specifically about genetic diseases (not predispositions), i.e. 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 hospitalized as a result of genetic disorders. Such conclusions were led not only by the rapid development of molecular genetics and the capabilities of genetic analysis, but also by the emergence of new neuroimaging methods, such as MRI. Using MRI, it is possible to determine damage to which area of ​​the brain leads to a disorder that occurs in a child, and often when we suspect a birth injury, we detect changes in structures that could not have been damaged during childbirth, and then an assumption arises about the genetic nature of the disease, about improper formation of organs . According to the results of recent studies, the influence of even difficult births with intact 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 death sentence, but a way to find the right path to treatment and correction of the disorder. Many diseases today are treated successfully, for others geneticists can offer more effective ways therapies that significantly improve the child’s quality of life. Of course, there are also disorders that doctors cannot yet overcome, but science does not stand still, and new treatment methods appear every day.

In my practice there was one very typical case. An 11-year-old child consulted a neurologist about cerebral palsy. Upon examination and questioning of relatives, suspicions arose about the genetic nature of the disease, which was confirmed. Fortunately for this child, the identified disease can be treated even at this age, and by changing 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 best known example is phenylketonuria. It manifests itself as developmental delay, mental retardation. If a diet without phenylalanine is prescribed in a timely manner, the child will grow up completely healthy, and after 20 years, the severity of the diet can be reduced. (If you give birth in a maternity hospital or medical center, your baby will be tested for phenylketonuria in the first days of life).

The number of such diseases has increased significantly. Leucinosis also belongs to the group of metabolic diseases. With this disease, treatment should be prescribed during the first months of life (it is very important not to be late), since the toxic products of impaired metabolism lead to faster damage to nervous tissue than with phenylketonuria. Unfortunately, if the disease is detected 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 so 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 disease, Grover disease, Felidbacher disease, Rett 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 disorders, and not just to compensate for them, which in many cases allows you to achieve serious success and even cure the baby.

What symptoms may indicate the genetic nature of the disease?

First of all, this is a child’s developmental delay, including intrauterine (from 50 to 70% according to some estimates), myopathies, autism, which cannot be treated epileptic seizures, any malformations of internal organs. The cause of cerebral palsy can also be genetic disorders; usually in such cases, doctors talk about the atypical course of the disease. If your doctor recommends undergoing genetic testing, do not delay it, in this case time is very valuable. Missed pregnancies and recurrent miscarriages, including among relatives, may also indicate the possibility of genetic abnormalities. It is very disappointing when the disease is detected too late and can no longer be corrected.

If the disease has no cure, do parents need to know about it?

Knowledge about the genetic nature of the disease in a child allows you to avoid the appearance of other sick children in this family. This is probably the main reason why it is worth undergoing genetic counseling at the stage of pregnancy planning, if one of the children has developmental defects or serious illnesses. Modern science makes it possible to carry out both prenatal and preimplantation genetic diagnostics, if there is information about a disease for which there is a risk of occurrence. At this stage, it is not possible to immediately test for all possible genetic diseases. Even healthy families, in which both parents have never heard of any disease, are not immune from the appearance of children with genetic abnormalities. Recessive genes can be passed on through dozens of generations and it is in your couple that you will meet your other half (see picture).

Should you always consult a geneticist?

You need to undergo genetic testing based on the presence of a problem if you or your doctor have any suspicions. No need to examine healthy child just in case. Many people say that they went through all the screenings during pregnancy and everything was fine, but here... In this case, you need to understand that screening examinations are aimed at identifying (and very effectively) the most common genetic diseases - Down, Patau and Edwards diseases, mutations in individual genes discussed above are not detected during such an examination.

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 training. We also see a geneticist who specializes in pregnancy problems. The advantage of a paid center is the doctor’s ability to devote more time to his patient (the appointment lasts two hours, and the search for a solution to the problem usually continues even after). There is no need to be afraid of a geneticist, he is simply a specialist who can make a diagnosis that allows him to cure a seemingly hopeless disease.

“Health Magazine for Expectant Parents”, No. 3 (7), 2014

Genetics in Israel is developing rapidly, and progressive methods for diagnosing and treating hereditary diseases are appearing. The range of specialized research is constantly expanding, the laboratory base is increasing and medical personnel are improving their qualifications. The ability to make a diagnosis as early as possible and begin comprehensive treatment of hereditary disorders makes treatment for children in Israel the most popular 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, Tel Aviv specialists medical center named after Sourasky (Ichilov Clinic) successfully conduct diagnostics, make an accurate diagnosis and provide comprehensive recommendations on the further treatment plan.

It should be understood that if radical intervention is not possible, 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. Relieving symptoms, mapping out further actions and predicting subsequent changes in health - all this is possible after diagnosis accurate diagnosis. You can promptly undergo examination and confirm the presence of a genetic disorder at the Ichilov clinic, after which the patient will be prescribed comprehensive treatment for the identified disease.

The Sourasky Center offers testing and examination not only for children, but 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 likelihood of the birth of healthy offspring, after which the doctor will determine further treatment 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 disorders are prescribed 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 disorders; 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 with leading Israeli specialists help to identify the presence of abnormalities at an early stage and begin treatment in a timely manner.

This includes the following diseases of children:

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

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

For early diagnosis of hereditary defects in children at the Ichilov Medical Center they use various methods laboratory research:

• molecular, which makes it possible to establish a deviation in DNA at the stage of intrauterine development of the fetus;
• cytogenetic, in which chromosomes are examined in various tissues;
• biochemical, which determines metabolic abnormalities in the body;
• clinical, helping to establish the causes of occurrence, 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 make a primary diagnosis. Parents will be offered the most innovative methods of technological development to determine genetic mutations.

In total, science currently knows 600 genetic abnormalities, so timely screening of a child will allow the disease to be identified and proper treatment to begin. Genetic testing a newborn is one of the reasons why women prefer to give birth at the Ichilov Clinic (Suraski).

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

Genetic diseases have very heterogeneous characteristics, so treatment is prescribed taking into account the clinical manifestations and individual parameters of the patient. In many cases, inpatient treatment is preferred. Doctors should have the opportunity to conduct the most extensive examination of a small patient, select drug regimen, if indicated, perform surgery.

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 and depends 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 symptoms of the disease, diet and physiotherapy.

The main directions of the treatment process at the Sourasky Center

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

• The etiological method is the most effective, aimed at the causes of health problems. The newest method of gene correction involves isolating a damaged piece of DNA, cloning it, and introducing a healthy component in its original place. This is the most promising and innovative method of combating hereditary health problems. Today, the task is considered extremely difficult, but is already used for a number of indications.
• The pathogenetic method affects internal processes occurring in the body. In this case, the pathological genome is affected, the physiological and biochemical state of the patient is adjusted by all available means.
• The symptomatic method of influence is aimed at relieving pain, negative conditions and creating obstacles for further development diseases. This direction is used independently 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 drugs to alleviate the manifestations of diseases. These are anticonvulsants, painkillers, sedatives and other drugs that should be given to a child only after a medical prescription.
• A surgical method is sometimes necessary to correct external defects and internal anomalies of the child’s body. Indications for surgical intervention are prescribed extremely carefully. Sometimes a lengthy preliminary examination and treatment is required to prepare a small patient for surgery.

As a positive example of the treatment of children in Israel, we can cite statistics on a common genetic disease - autism. At the Ichilov-Suraski hospital early detection anomalies (from six months of life) enabled 47% of such children to develop normally in the future. Doctors considered the detected disorders in the remaining children examined to be insignificant and not requiring therapeutic intervention.

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

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