A brief encyclopedia of gene therapy

Gene therapy, or how to drive DNA into a cell and cure patients with it

Azat Murtazin, "Nanometer"

Introduction

Every year, more and more articles about medical clinical trials appear in scientific journals, in which, one way or another, treatment based on the introduction of various genes was used – gene therapy. 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 caused by a defect in a single gene, as well as many others [1]. Or, for example, gene therapy can help out and save a limb for those patients who have narrowed the lumen of the vessels in the lower extremities and, as a result, developed persistent ischemia of the surrounding tissues, that is, these tissues experience a severe lack of nutrients and oxygen, which are normally carried by blood throughout the body [2]. Surgical manipulations and medications often fail to treat such patients, but if the cells are 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 it would become much easier for patients to live.

Gene therapy today can be defined as the treatment of diseases by introducing genes into patients' cells in order to change gene defects or give cells new functions. The first clinical trials of gene therapy methods were undertaken quite recently – on May 22, 1989 in order to diagnose cancer. The first hereditary disease for which gene therapy methods were applied was hereditary immunodeficiency [3].

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 [4].

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 (Lat. literally "in the living") have not been studied enough. In countries with the most advanced level of research in this field, especially in the USA, medical protocols using semantic DNA sequences are subject to mandatory examination in the relevant committees and commissions. In the USA, these are the Advisory Committee on Recombinant DNA (Recombinant DNA Advisory Committee, RAC) and the Food and Drug Administration (FDA), followed by mandatory approval of the project by the Director of the National Institutes of Health [3].

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

Cell transfection

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

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

1. The point of origin of replication (ori) is the sequence of nucleotides from which DNA doubling begins. If the vector DNA cannot be doubled (replicated), then the necessary therapeutic effect will not be achieved, because it will simply be quickly split by intracellular enzymes-nucleases, and due to the lack of matrices, much fewer protein molecules will eventually be formed. It should be noted that these points are specific to each biological species, that is, if vector DNA is supposed to be obtained by multiplying it in bacterial culture (and not just by chemical synthesis, which is usually much more expensive), then two separate points of replication start will be required – for humans and for bacteria;

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

Formation of "sticky ends" involving restrictases

These sites are necessary in order to stitch vector DNA (which, in fact, is a "dummy") with the necessary therapeutic genes into a single molecule. Such a molecule sewn from two or more parts is called "recombinant";

3. It is clear that we would like to get millions of copies of a recombinant DNA molecule. Again, if we are dealing with a culture of bacterial cells, then this DNA needs to be isolated further. The problem is that not all bacteria will swallow the molecule we need, some will not do it. In order to distinguish these two groups, selective markers are inserted into the vector DNA - sites of resistance to certain chemicals; now if these very substances are added to the medium, 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).

The very process of introducing a plasmid vector into certain cells is called transfection. A plasmid is a rather short and usually circular DNA molecule that is located 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, transmit resistance to certain antibiotics.

Plasmids exist in bacteria under natural conditions. But no one can prevent a researcher from artificially synthesizing a plasmid that will have the properties necessary for him, sewing a gene insert into it and embedding it into a cell. Different inserts can be sewn into the same plasmid [5].

Methods of gene therapy

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

1. Fetal, in which foreign DNA is injected into a zygote (fertilized egg) or embryo at an early stage of development; it is expected that the injected material will get into all recipient cells (and even into germ cells, thereby ensuring transmission to the next generation). In our country, it is actually banned [6];

2. Somatic, in which genetic material is injected into non-reproductive cells already born and it is not transmitted to the germ cells.

In vivo gene therapy 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 injectable vaccines. Aerosol gene therapy is being developed, as a rule, for the treatment of lung diseases (cystic fibrosis, lung cancer).

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

It is important that when drawing up gene correction schemes, the effectiveness of transfection, the degree of correction of the primary biochemical defect in cell cultures (in vitro, "in vitro") and, most importantly, in vivo on animal biological models are evaluated. Only after that it is possible to start the program of clinical trials [7].

Direct delivery and cellular carriers of therapeutic genes

There are many methods of introducing foreign DNA into a eukaryotic cell: some depend on physical processing (electroporation, magnetofection, etc.), others depend on the use of chemical materials or biological particles (for example, viruses) that are used as carriers. It is worth mentioning right away that chemical and physical methods are usually combined (for example, 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 the use of 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. The disadvantage is that the nucleus reaches only about 1 – 10% of the DNA. The method is used in vitro to transfer DNA into human cells (Fig.3);

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

     c) A very effective method for DNA transfection is its introduction through liposomes – small, surrounded by a membrane of the corpuscle, which can merge 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 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 effectiveness of gene transfer using liposomes is limited, since the DNA introduced by them in cells is usually immediately captured by lysosomes and destroyed. The introduction of DNA into human cells using liposomes is now the main thing in in vivo therapy (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 penetrates into the cell by endocytosis. DEAE-dextran changes 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 been widely used in gene therapy;

     e) With the help of histones and other nuclear proteins [8]. These proteins, which contain many positively charged amino acids (Lys, Arg), naturally help to compactly lay a long DNA chain in a relatively small cell nucleus.

2. Physical methods

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

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

     c) Sonoporation is a method of artificial transfer of foreign DNA into cells by exposing them to ultrasound, causing the opening of pores in the cell membrane;

     d) Optical transfection is a method in which a tiny hole is made in the membrane (about 1 microns in diameter) using a highly focused laser;

     e) Hydrodynamic transfection is a method of delivering genetic constructs, proteins, etc. by a controlled increase in pressure in 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 the delivery is non-specific. The delivery efficiency for skeletal muscle is from 22 to 60% [9];

     e) DNA microinjection – introduction into the nucleus of an animal cell 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 is a gene gun, while DNA is bound into a nanoparticle with inert solids (more often gold, tungsten), which then "shoots" directly into the nuclei of target cells. This method is used in vitro and in vivo to introduce genes, in particular, into muscle tissue cells, for example, in a disease such as Duchenne myodystrophy. The sizes of gold particles are 1-3 microns (Fig. 7).

     b) Magnetofection is a method that uses the forces of magnetism to deliver DNA to target cells. First, nucleic acids (NC) 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%, obvious non-toxicity is noted. After 10-15 minutes, the particles are registered in the cell – this is much faster than other techniques. 

     c) Impalefection (impalefection; "impalement", lit. "impaling" + "infection") is a delivery method using nanomaterials, such as carbon nanotubes and nanofibers. In this case, the cells are literally pierced with a litter of nanofibrils [10]. The prefix "nano" is used to denote their very small sizes (within billionths of a meter) (Fig.8).

Separately, it is worth highlighting such a method as RNA transfection: it is not DNA that is delivered to the cell, but RNA molecules - their "successors" in the chain of protein biosynthesis; at the same time, special proteins are activated that cut RNA into short fragments – the so-called small interfering RNAs (miRNAs). These fragments bind to other proteins and, eventually, this leads to inhibition of the expression of the corresponding genes by the cell. In this way, it is possible to block the action of those genes in the cell that are potentially doing 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 considered. Now we can proceed to the consideration of viral methods. Viruses are non–cellular forms of life, most often representing a molecule of nucleic acid (DNA or RNA) wrapped in a protein envelope. If you cut out from the genetic material of the virus all those sequences that cause the occurrence of diseases, then the whole 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 adenoassociated viruses (AAV) are most often used. To begin with, it is worth figuring out what the ideal candidate for transduction among viruses should be. The criteria are such that it should be:

• stable;
• emok, that is, to contain a sufficient amount of DNA;
• it is inert in relation to the metabolic pathways of the cell;
• accurate – ideally, it should embed its genome into a specific locus of the host 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).

Of all the three listed most used viruses, AAV is the safest and at the same time the most accurate. Their almost only drawback is a relatively small capacity (approx. 4800 bp), which, however, turns out to be sufficient for many genes [5].

In addition to these methods, gene therapy is often used in combination with cellular therapy: at the same time, a culture of certain human cells is first planted in the nutrient medium, after that the necessary genes are introduced into the cells in one way or another, cultured for some time and transplanted back into the host body. As a result, the cells can be restored to their normal properties. So, for example, human white blood cells (leukocytes) were modified in leukemia (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, then let's focus 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 reach the cell nucleus so that a special enzyme – RNA polymerase - synthesizes a molecule of informational RNA (mRNA) on the 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, vascular growth factor (VEGF), which will begin to perform a certain therapeutic function (in this case, it will start the process of formation of vascular branches in tissue susceptible to ischemia).

As for the expression of the introduced genes in the required cell type, this task is solved with the help of regulatory elements of transcription. The tissue in which expression occurs is often determined by a combination of an enhancer specific to this tissue (an "amplifying" sequence) with a specific promoter (a sequence of nucleotides from which RNA polymerase begins synthesis), which can be induced [11, 12]. It is known that gene activity can be modulated in vivo by external signals, and since enhancers can work with any gene, insulators can also be introduced into vectors that help the enhancer work regardless of its position and can behave as functional barriers between genes. Each enhancer contains a set of binding sites of activating or suppressing protein factors [11]. Promoters can also be used to regulate the level of gene expression. For example, there are metallothioneine or temperature-sensitive promoters; promoters controlled by hormones.

The expression of a gene depends on its position in the genome. In most cases, existing viral methods lead only to the random embedding of a gene into the genome. To eliminate such dependence, when constructing vectors, the gene is supplied with known nucleotide sequences that allow the gene to be expressed regardless of where it is embedded in 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 can be useful in some situations, such as the implementation of 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), insect steroid, ecdysone or its analogues, the anti-progestin drug mayfpriston (RU486) and chemical dimerizers, such as rapamycin and its analogues. All of them involve drug-dependent attraction of the transcription activation domain to the main promoter leading the desired gene, but differ in the mechanisms of this attraction [13].

Conclusion

A review of the data allows us to conclude that, despite the efforts of many laboratories around the world, all vector systems already known and tested in vivo and in vitro are far from perfect [14,15]. If the problem of delivering foreign DNA in vitro is practically solved, and its delivery to target cells of different tissues in vivo is successfully solved (mainly by creating structures carrying receptor proteins, including antigens specific to certain tissues), then other characteristics of existing vector systems are integration stability, regulated expression, security – still in need of serious improvements.

First of all, it concerns the stability of integration. Until now, integration into the genome has been achieved only with the use of retroviral or adeno-associated vectors. It is possible to increase the efficiency of stable integration by improving gene constructs such as receptor-mediated systems or by creating sufficiently stable episomal vectors (that is, DNA structures capable of staying inside nuclei for a long time). Recently, special attention has been paid to the creation of vectors based on artificial mammalian 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-sized (genomic) genes and their natural regulatory elements, which are necessary for the proper 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 of organs, which will undoubtedly significantly expand the arsenal of methods for biomedical research and create new opportunities for the preservation and prolongation of human life [16].

References:

1. Stefano Ferrari E. A., Griesenbach U. Progress and Prospects: Gene Therapy Clinical Trials (Part 1). Gene Therapy (2007) 14, 1439–1447; doi:10.1038/sj.gt.3303001
2. Plotnikov M.V., Rizvanov A.A., Masgutov R.F., Mavlikeev M.O. The first clinical experience of using direct gene therapy of VEGF and bFGF in the treatment of patients with critical lower limb ischemia. Cell Transplantology and Tissue Engineering Volume VII, No. 3, 2012
3. Culver K.W. Gene Therapy: A Handbook for Physicians. N.Y.: May Ann Liebert Inc. Publ., 1994. 117 p.
4. Torrecilla J. et al. Lipid Nanoparticles as Carriers for RNAi against Viral Infections: Current Status and Future Perspectives. BioMed Research International
Volume 2014 (2014), Article ID 161794, 17 pages
5. Bashmakova V. Molecular cloning, or How to put foreign genetic material into a cell 6. Federal Law "On State Regulation in the field of genetic engineering activities" dated 5.06.1997 (as amended.
Federal Laws of 12.07.2000 N 96-FZ, of 30.12.2008 N 313-FZ, of 04.10.2010 N 262-FZ)
7. Baranov V.S. Gene therapy -- medicine of the XXI century. Soros Educational Journal, No. 3, 1999. pp. 63-68.
8. Solovyova V.V., Kudryashova N.V., Rizvanov A.A. Transfer of recombinant nucleic acids into cells (transfection) using histones and other nuclear proteins. Cell Transplantology and Tissue Engineering 2011; VI(3):29-40.
9. Goigorian A.S., Shevchenko K.G. Possible molecular mechanisms of functioning of plasmid structures containing the VEGF gene. Cell Transplantation and Tissue Engineering 2011; VI(3):24-28.
10. http://en.wikipedia.org/wiki/Transfection 11. Sverdlov E.D. Essays on modern molecular genetics.
Essay 5. Transgenosis and new molecular genetics // Molek. genetics, microbiol., virosol. - 1996. - No. 4. - pp. 3-32.
12. Sverdlov E.D. Essays on modern molecular genetics. Essay 6. Gene therapy and medicine of the XXI century // Molek. genetics, microbiol., virosol. - 1997. - No. 2. - pp. 3-28.
13. Clackson T. Regulated gene expression systems // Gene Therapy. - 2000. - Vol. 7. - P. 120-125.
14. Hodgson C.P. The Vector Void in Gene Therapy // BioTechnology. 1995. Vol. 13. P. 222-225.
15. Smith K.T., Stepherd A.J., Boyd J.E., Lees J.M. Gene Delivery Systems for Use in Gene Therapy: An Overview of Quality Assurance and Safety Issues // Gene Therapy. 1996. Vol. 3. P. 190-200.
16. Tkachuk V.A. Gene and cell therapy in modern biology and medicine. Abstracts, 9.03.2010.

Portal "Eternal youth" http://vechnayamolodost.ru 02.02.2015