RU2372941C2 - Pharmaceutical composition for genetic therapy of diseases requiring stimulation of regenerative processes, including human tissue damages of various aetiologies, based on synthetic modified gene of insulin-like human growth factor type one (ifr-1, igf-1) - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to genetic therapy and concerns nucleotide sequence that codes insulin-like human growth factor IGF-1 presented with a synthetic gene including a sequence SEQ ID NO:1, recombinant plasmid DNA, containing this sequence, an eukaryotic cell containing recombinant plasmid DNA, construction for genetic therapy and a pharmaceutical composition for genetic therapy with regenerative and wound healing action.

EFFECT: advantage of the invention consists in decreased doses and introduction rate of injected preparations.

5 cl, 2 ex, 2 tbl, 4 dwg

Description

FIELD OF THE INVENTION

The invention relates to the field of medicine, pharmacology, biotechnology, genetic engineering, gene therapy, and in particular to the treatment of diseases requiring stimulation of regenerative processes, including damage to human tissues of various etiologies, using a pharmaceutical composition or preparation for gene therapy based on a synthetic modified gene of insulin-like factor human growth of the first type.

The present invention also relates to the use of a vector for gene therapy carrying a synthetic modified gene of human insulin-like growth factor.

State of the art

Peptide growth factors are a group of low molecular weight proteins that affect the growth and function of individual cells and body tissues. Common functions of peptide growth factors include: stimulation of mitosis, influence on the processes of differentiation and growth of cells, participation in the processes of embryogenesis, tissue regeneration.

Regeneration processes are one of the most important mechanisms for maintaining the homeostasis of the body, especially with injuries of various etiologies. These include mechanical injuries, including operating, chemical, thermal, radiation, ischemic, and others. The regeneration of damaged tissues depends mainly on proliferation in the affected area of cells of various types and the synthesis by cells of both their own elements and components of the intercellular substance.

Accelerating wound healing and treating other injuries by stimulating regeneration is one of the most important tasks of practical medicine. Various regeneration agents are used as regeneration stimulants: 2'-deoxyribonucleotides (patent No. 215148), liposomes containing alpha-tocopherol (patent No. 2036637), avocado oil and mineral particles (patent No. 2173982), fetal cartilage introduced intraarticularly for regeneration of the articular cartilage (patent No. 2195940), a bio-antioxidant solution of thiophan based on an oil solution of alpha-tocopherol (patent No. 2297217), extracts of housefly larvae for skin rejuvenation with a group of minerals (patent No. 2289412), physiological saline solution (pat nt No. 2053775), a mixture of vitamins with extract of extract of the bark of a pork or fetal adrenal gland of a person (patent No. 2185835), simultaneous irradiation with high-frequency fields and low-intensity quasi-monochromatic radiation of the visible and near infrared range (patent No. 2252516), local administration of hyaluronic acid (patent No. 28) , donated blood and its components (patent No. 2191585), a mixture of sunflower oil, sea buckthorn oil, lavender oil (No. 2097017), quidine ammonium salt of conidine (patent No. 2020933), 5- (2-pyrazinyl) -4-methyl-1, 2-dithiol-3thion (pa awning No. 2291696), synthetic peptides for liver regeneration (patents No. 2297239, No. 2286794), neurotrophic factors AF-1 and AF-2 (patent No. 2157223), conditioned culture medium obtained by culturing eukaryotic cells in three dimensions (patent No. 2280459 ) The regenerative effect of most of these agents and substances is associated either with an indirect or trophic effect. That is why many peptide growth factors that act as direct mitogens can be considered as the most promising drugs. They can be used as part of various biological extracts (for example, from the placenta) or by obtaining recombinant proteins using producers based on various organisms. These include various bacterial producers, yeast, and other eukaryotic cells.

However, when using recombinant proteins, there are a number of disadvantages. In particular, frequent introduction of proteins is required due to their rapid biodegradation in the body, significant fluctuations in the level of the introduced protein in the body occur, recombinant proteins may not have natural glycosylation and other post-translational modifications, purification of recombinant proteins requires the use of complex methods of purification, refolding.

In this sense, the use of gene therapy when using various vectors carrying the genes of growth factors and the corresponding regulatory elements is a more promising direction. Compared with the use of recombinant proteins, gene therapy has a number of significant advantages:

- when applying gene therapy, frequent injections of the drug are not required, since the expression of target genes in the patient's body can continue, depending on the type of construct used and the goals of therapy, from several days to many years and even for life;

- the levels of the target protein formed in the body are more constant, no significant fluctuations are observed;

- human proteins formed with the help of vectors in the patient’s cells do not differ completely from natural ones, since all stages of natural post-translational changes undergo;

- purification of drugs for gene therapy as a whole is simpler and easier can be standardized;

- no refolding of therapeutic constructs is required, unlike recombinant proteins produced by producers.

For gene therapy, a number of vectors, primarily viral, are used. However, the latter have a number of disadvantages, such as the emerging immune response to a viral vector (for example, an adenovirus), the risk of tumor transformation (retroviral vectors), etc. In this sense, plasmids are the safest vectors. One of the most serious problems of gene therapy when using plasmids as vectors is the unresolved issue of their highly efficient delivery to cells, which often determines the level of gene expression and synthesis of the target protein insufficient to achieve a therapeutic effect.

Gene therapy can be used to treat wounds. So, a DNA molecule, included in the biocompatible matrix and carrying any gene product, can be delivered to repair cells in the wound area (patent No. 2170104). Among these genes are considered, including various growth factors, including insulin-like growth factor (the type of this factor is not indicated).

The insulin-like human growth factor of the first type (IGF-1, IGF-1) is a peptide growth factor mediating the growth-promoting effects of growth hormone (STH). It is a polypeptide consisting of 153 amino acids, with a molecular weight of approximately 17 kDa. Under the influence of STH, IGF-1 is formed in the liver and other tissues. In turn, IGF-1 inhibits the secretion of STH by a negative feedback mechanism.

IGF-1 is present in the blood mainly in complex with binding proteins that modulate the action of insulin-like growth factors (Clemmons D.R. et al., 1993; Krywicki R.F., 1992). He is involved in fetal development and postembryonic development. IGF-1 stimulates the proliferation of cells in most tissues, especially bone, cartilage and muscle. Along with growth hormone, IGF-1 determines the growth of a person and the size of his organs (Kahn C.R. et al., 1981).

In vitro IGF-1 is able to induce metabolic effects that are fundamentally similar to insulin, realizing them through cross-interaction with insulin receptors (Le Roith D. et al., 1993). Insulin receptors and IRF-1 receptors are transmembrane glycoproteins (class tyrosine protein kinases) of a tetrameric structure (two extracellular ligand-binding alpha subunits and two transmembrane effector beta subunits with a tyrosine kinase cytoplasmic domain) stabilized by two species. Both protein kinase receptors have a similar principle of hormonal signal transmission: tyrosine phosphorylation of the cascade of mediating proteins under the action of the beta kinase subunit protein kinase, which, after binding of the corresponding peptide ligand to the extracellular alpha subunit, is activated by autophosphorylation (Le Roith et al. 1993). IRF-1 is an anabolic agent that has been shown to improve metabolism (Meyer NA et al., 1996) [35], intestinal mucosal function (Huang KF et al., 1993) [42] and decreased protein loss (Strock LL et al ., 1990) [43] after various types of damage. IRF-1 mediates growth hormone activity in the hypermetabolic state by attenuating loss of lean body mass, impaired immune response, acute phase response and by improving wound healing (Zaizen Y. et al., 1990; Clemmons DR 1994; Lo HC et al. , 1995; Guler et al., 1988; Walker JL et al., 1991) [35, 38, 44-47]. IRF-1 treatment improves tissue healing (Martin P.1997; Steenfos H. 1994; Pierre et al., 1997) [40, 41, 48]. IRF-1 was used to reduce the manifestations of toxic neuropathy (patent No. 2120803). There are adverse side effects such as hypoglycemia, changes in mental state, edema, fatigue and headaches that limit the therapeutic use of the IRF-1 peptide (Jabri N. et al., 1994) [49, 50]. These adverse side effects are caused by superphysiological doses of free IRF-1, which are necessary to achieve biological effectiveness and stimulate regeneration processes in a specific local place with systemic administration (Bondy S.A. et al., 1994) [49, 50]. In this regard, the development of a system for the local delivery of the IGF-1 peptide to the site of injury is an important condition for the full and safe treatment of traumatic injuries of various etiologies and the treatment of other diseases that require stimulation of regenerative processes. One solution to the problem is the use of gene therapy using various vectors carrying the target therapeutic gene. This design, which is included in the biocompatible matrix and carrying any gene product, including various growth factors, including insulin-like growth factor (the type of this factor is not indicated), can be used to treat wounds (patent No. 2170104).

Currently, the world is developing many systems for the delivery of DNA to target cells, such as liposomes or various viral systems. It was shown that with increasing delivery efficiency, its specificity decreases. So, liposomes carry DNA throughout the body and deliver it to various organs, including the heart, liver, spleen, lungs, etc. (Thierry A.R. et al., 1996). Viral vectors have several drawbacks - they are expensive and often have limited cloning capacity, which does not allow to regulate the expression of the "therapeutic gene" using tissue-specific sequences. In addition, viral proteins can cause an inflammatory reaction, which excludes the repeated administration of the vector. There are other dangers of using viral vectors.

Plasmid DNA based vectors that do not induce an immune response are free of such drawbacks. It was shown that during intramuscular injection of ordinary, “naked” plasmid DNA, it encounters a diffusion tissue barrier and transforms only the cells located near the injection site. Of course, the magnitude of this diffusion barrier depends on the state of the tissues and the intercellular matrix, but there is no significant distribution beyond the injection site throughout the body (Jiao et.al., 1990; Davis et.al, 1993).

Despite these advantages, one of the disadvantages of using naked plasmid DNA is the relatively low frequency of transformation of tissue cells at the injection site. Of the total number of introduced plasmid DNA, 0.01-1% of plasmids are involved in the process of cell transformation. Thus, the active part of the dose of such a drug is small. One way to solve this problem is to increase the expression of genes that make up the therapeutic plasmid. With this approach, despite the low level of transformation of cells of damaged and adjacent tissue damage zone, it is possible to achieve the necessary therapeutic effect without increasing the dose of the drug.

Modulation of gene expression is such a problem as the effect of the nucleotide composition of genes on their expression (Duret, 2000; Naya et.al., 2001). The effect of differences in codon composition on the stability of messenger RNA, its processing, and nucleocytoplasmic transport in various cellular systems is currently being studied (Graf et.al., 2000; Koresawa et.al., 2000; Liu et.al., 2002; Stratford et .al., 2000; Uchijima et.al., 1998; Yang et.al., 1996; Zhang et.al., 1999).

Disclosure of invention

The above disadvantages of viral vectors led to the inclusion in the basis of our developed pharmacological composition as a plasmid vector (naked DNA). This is the most secure vector for gene therapy.

In addition, limiting the spread of the plasmid throughout the body from the point of injection into the tissue due to the tissue diffusion barrier, which is a drawback from the point of view of gene therapy of the whole organism, is at the same time an advantage, if necessary, of local stimulation of regenerative processes. Such a local action may be necessary in various diseases, in particular, with tissue damage. This very important property also testified in favor of the inclusion of “naked” plasmid DNA (and not as part of liposomes or viral delivery systems) in the preparation we develop for local tissue regeneration. Thus, an important element of our invention is the local action of the IGF-1 factor and the absence of its significant effect on the body as a whole, which avoids the above-mentioned adverse effects that occur when using protein forms of the IGF-1 preparation.

One of the possible solutions to the problem of low efficiency of gene therapy based on plasmid constructs due to the difficulty of delivering a sufficient amount of them to cells is to increase the expression of target genes in the cell. This effect of a significant increase in the expression of target genes in the cell during gene therapy can be achieved in various ways, in particular, by changing the regulatory elements of the vector, as well as by creating synthetic genes. When using synthetic genes, their expression in the patient's cells can be significantly increased without changing the primary structure of the therapeutic protein.

We found that the matrix RNA of the IGF-1 gene, when its nucleotide composition changes, becomes more stable in mammalian cells and human cell culture. Based on the obtained data, we developed a modified IGF-1 gene (FGsintIFR-1, PGsyntIGF-1) with an optimized nucleotide composition without changing the amino acid sequence of the translated protein and synthesized it (see the list of sequences).

This synthetic gene has been cloned into recombinant plasmid DNA for eukaryotic expression. The nucleotide sequence of the synthetic gene FGsintIFR-1 provided an increase in the stability of mRNA in mammalian cells, which allowed using standard plasmid vectors for eukaryotic expression in human cells to increase the level of translation of the encoded protein up to 100 or more times compared to using a native, natural gene under similar conditions.

The drug we developed for gene therapy, or the pharmaceutical composition that includes it, is plasmid DNA (pC) that carries the modified synthetic gene FGsintIFR-1, which encodes and expresses the peptide growth factor IGF-1 in the human body (see the materials explaining the invention, FIG. .one).

We have shown that when using the synthetic gene FGsintIFR-1 with an optimized nucleotide sequence for gene therapy, lower amounts of plasmid DNA can be introduced, from 4 to 100 times smaller than the systems described in other patents No. 205114367, 200110435, 2001129537, 2004129332 and etc. This makes the use of a pharmacological preparation based on the plasmid DNA developed by us more economical, effective and safe.

The identity of the formed peptide to natural IGF-1 during expression of the synthetic gene and subsequent translation ensures the safety and effectiveness of the drug in terms of using the natural mechanisms of its metabolism and catabolism without the formation of toxic products. This property distinguishes this drug from both conventional chemical pharmaceuticals and protein preparations obtained using in vitro prokaryotic or eukaryotic producers. The reason is that in the first case, when using prokaryotic producers, many natural mechanisms of folding (folding) of proteins and their post-translational modifications do not work. In the second case, when using any eukaryotic producers (with the exception of human cells), the processes of folding and post-translational modifications may differ from the processes occurring in human cells. Using human cells as a producer is dangerous from the point of view of the spread of viruses, including oncogenic ones. In addition, the use of this drug for gene therapy, providing synthesis of the target polypeptide in vivo, will avoid the difficulties of production and protein purification processes in vitro. The production, purification and storage of DNA preparations is economically more profitable than protein ones, since the former are much more stable and can be produced in large quantities and at lower cost, and the purification of plasmid DNA is simpler than protein. Moreover, in the manufacture of a preparation based on plasmid DNA, unlike protein preparations, the folding steps are not required.

Thus, the developed design was used in the pharmaceutical composition as a drug for gene therapy of diseases of various etiologies that require stimulation of regenerative processes in a wide range of human tissues, including muscle, bone, all types of connective, epidermal, cartilage and nervous tissue.

Brief Description of the Drawings

(see materials explaining the essence of the invention).

Figure 1 - plasmid DNA pC carrying the modified synthetic gene FGsintIFR-1, encoding and expressing in the human body peptide growth factor IGF-1 (plasmid pCIGF-1).

Figure 2 - scheme for obtaining plasmids pCIGF-1.

Figure 3 - Western blotting of the lysate of 293T cells transformed with plasmids pCIGF-1 (2nd lane), pCIGF-Isynt (3rd lane) carrying the native gene IGF-1 and FGintynt-1, respectively. Lane 1 — lysate of non-transformed cells; 4th lane — protein marker (Fermentas, Lithuania).

Figure 4 - the level of IGF-1 protein in the culture medium of 293T cells transformed with plasmids expressing native IGF-1 (IGF-1) and synthetic modified FGSintIFR-1 (IGF-1 synt) genes.

Figure 5 - timing of epithelialization of full-layer skin wounds depending on the plasmid DNA used.

6 is a change in the area of granulating wounds in the treatment process.

The implementation of the invention

The synthesis of the FGsintIFR-1 gene was carried out by lengthening mutually overlapping oligonucleotides (primers) according to the described methods (Majumder, 1992). Oligonucleotides were fragments of a gene with a length of about 60-70 nucleotides with mutually overlapping sections with a length of about 20 nucleotides. The main requirements for primers were that their length should not exceed 70 nucleotides, and the hybridization sites should be at least 20 nucleotides. In addition, the ends of the oligonucleotides should not have long sections with repeating G or C. In some cases, the selection of optimal primers was carried out empirically, by shifting the primer relative to the matrix or changing the length of the primer by 3-6 nucleotides. In total, 36 primers were used to synthesize a gene with a length of 462 nucleotides. The synthesized fragments with a length of about 100 nucleotides were isolated by gel electrophoresis and cloned into the pGEM-T Easy plasmid vector. Cloning was carried out using the restriction sites Kpnl, SacII, EcoRV, BamHI or through “blunt” ends. After sequencing, the fragments were cut from the vector using the appropriate restriction enzymes or amplified, after which they were combined into the nucleotide sequence of IGF-1 by fusion using the polymerase chain reaction (PCR) After the final step of synthesizing FGsintIFR-1 by ligation of the fragments, the artificial gene was cloned into the pGEM-T vector at the restriction sites Kpnl and Sacl. The synthetic gene was fl it is surrounded by additional EcoRI restriction sites at the 5'-end and Xhol at the 3'-end. Next, the artificial gene was cloned into the pC expression vector under the control of the cytomegallovirus promoter. The pC vector is designed for gene expression in eukaryotic cells. It contains the basic regulatory elements: the early promoter cytomegalovirus proteins, site for termination of transcription, polyadenylation and origin of ColEl replication from the high copy plasmid pBR322. This vector was designed by Pharma Gene LLC by the authors of this patent. The scheme for obtaining the plasmid is presented in the drawing (see materials explaining the essence of the invention, figure 2).

For cloning, the sequence of the FGsintIFR-1 gene (462 nucleotides) was isolated from the gel and subsidized into the pC expression vector at the Xhol and EcoRI restriction sites. The ligation reaction was carried out using the DNA Ligation Kit (Sigma, USA) under the following conditions: ligation buffer (25 mM Tris-HCl pH 7.8; 10 mM MgCl ₂ ; 1 mM DTT; 0.5 mM ATP), 400 ng of prepared fragments, 100 ng of restricted vector DNA, 1 unit Phage T4 DNA ligases. The reaction was carried out for 8 hours at 16 ° C. The ligase mixture was dialyzed on nitrocellulose filters (Millipore) using dialysis buffer (glycerol 0.5%; EDTA 5 tM).

The ligase mixture was transformed using thermal Ca2 ++ - transformation into bacterial cells. We used cell E.coli DH10B / R (Gibko BRL, USA) with genotype ^{F - mcrA Δ (mrr-hsdRMS} -mcrBC) φ80dlacZΔM 15 ΔlacX74 deoR recA1 endA1 araD139 Δ (ara , leu) 769 galU galKλ ^- rpsL nupG. To prepare competent cells, a separate colony was grown on LB agape (Gibko BRL, USA) in a Petri dish and placed in 5 ml of LB medium (Gibko BRL, USA). Cells were grown for eight hours at 37 ° C under continuous stirring (250 rpm). 2 ml of the resulting culture was placed in 200 ml of LB medium. Cells were grown at 37 ° C with constant stirring (250 rpm) to an OD of ₆₀₀ = 0.6, after which they were besieged by centrifugation for 10 minutes at 4000g at 4 ° C. Cells were washed in deionized water, followed by centrifugation. The washing procedure was repeated three times. After washing, the cell pellet was resuspended in a small volume of deionized water and centrifuged for 30 sec at 12000 rpm in a microcentrifuge. To the cell pellet, 3 volumes (of the cell pellet volume) of 15% glycerol were added, the pellet was resuspended and quickly frozen in liquid nitrogen. The cells ready for transformation were stored at a temperature of -70 ° C.

Sowing transformed cells was performed on solid LB medium. For fast screening of clones containing a plasmid with a cloned insert, the PCR method was used. The reaction was carried out in a volume of 25 μl in thin-walled polypropylene tubes with a volume of 650 μl containing 10 mm Tris-HCl pH 8.3; 1.25 mM MgCl ₂ ; 50 mM KCl; 200 μM each of dNTP; 200 nM of each of the primers; 1.5-2 μg of cellular DNA and 2.5 units. Taq polymerase (Litech, Russia). The reaction mixture was heated for 1 minute at 95 ° C to denature the cell DNA. To prevent evaporation, 15 μl of Bayol F (Sigma, United States) was layered onto a 25 μl reaction mixture. The amplification reaction was carried out in the Tertsik thermal cycler (DNA technology, Russia) under the following conditions - 32 cycles: 10 seconds at 95 ° C; 15 seconds at T _lifespan (° C); 60 seconds at Testgesa 72 ° C. The biomass of one colony about 1 mm in size was used as a matrix. An additional cycle was completed to complete the formed DNA chains: 5 minutes at 72 ° C.

Three clones were selected. Plasmid DNA was isolated from the clones. Isolation of plasmid DNA was performed for restriction analysis of the studied plasmids, transformation of eukaryotic cells in order to analyze the expression of the IGF-1 gene, as well as in the analysis of recombinant clones for the presence of the desired insert. For mini-isolation of plasmid DNA, a separate colony grown on LB agar was taken and placed in 5-10 ml of LB medium with ampicillin (50 μg / ml). Cells were grown for eight hours at 37 ° C under constant stirring (250 rpm). The resulting culture of bacterial cells was besieged at 4000 g for 10 minutes at 4 ° C. The supernatant was discarded, and plasmid DNA was isolated from the cell pellet using the Wizard Minipreps DNA Purification System kit (Promega, USA) according to the instructions attached to the kit. The isolated plasmid DNA was analyzed by 0.8% agarose gel electrophoresis, its concentration was measured and stored at -20 ° C.

In the course of further work, a restriction analysis of the obtained plasmids was performed for the presence of a fragment containing the FGintyntr-1 gene.

The accumulated plasmid was used to transform 293T eukaryotic cells. The day before transformation, eukaryotic cells were reseeded at a density of 10 ⁴ / cm. The next day, the culture medium was changed to fresh, containing 10% fetal calf serum. After 3 hours, calcium phosphate DNA precipitate was added. For a 25 cm ² vial containing 5 ml of medium, the precipitate was prepared as follows: a solution containing 5 μg of DNA was added to one tube, then 31 μl of 2M CaCl ₂ and the total volume was adjusted to 0.25 ml with water; 0.25 ml of 2 × HBS (1.64% NaCl (by weight); 1.13% HEPES (by weight); 0.04% Na ₂ HPO ₄ (by weight) and pH 7.12) were added to another tube. The DNA solution was added dropwise to HBS. The precipitate was applied to the cells by slightly tilting the cup and adding it to the medium. Cells were incubated for 4 hours and washed with serum-free medium. To increase the efficiency of transformation at this stage, the cells were subjected to glycerin shock. For this, 0.5 ml of a 15% solution of glycerol in HBS was added to the cell vial and incubated at 37 ° C for 2 minutes. The shock solution was removed until the cells began to shrink. Then the cells were washed and poured with fresh medium. After 3 days, the cells were removed from the substrate and analysis was performed on the expression of transformed genes. The transfer of transformed eukaryotic cells proteins of the cell lysate of the transformed eukaryotic cells previously fractionated in the PAAG gel under denaturing conditions (Bio-Rad, USA) was carried out according to the Western blot method (Ngo T.T. et al., 1988) with some modifications. Before starting the transfer, the gel with fractionated 293T cell lysate proteins and the nitrocellulose membrane were soaked in transfer buffer (0.025 M Tris; 0.193 M glycine; 20% ethanol) for 10 minutes. Proteins were transferred from the gel by electrotransfer (electric field strength 7.5 V / cm) for 1.5 hours in a cell specially designed for this purpose (Bio-Rad, USA). After the transfer was completed, the blot was washed for 5 minutes in a solution of FSB-tween (0.01 M Na ₂ HPO ₄ / NaH ₂ PO ₄ pH 7.2; 0.15 M NaCl; 0.3% tween-20) at room temperature. The blot area corresponding to a mixture of standard proteins was stained with an amide black dye (Sigma, USA). The blot was dried at room temperature and stored in sealed bags at 4 ° C until proteins were detected. Monoclonal antibodies were used to detect the IGF-1 protein. To prevent non-specific binding of antibodies to the blot, non-specific binding sites were blocked with a 2.5% solution of Skim Milk (Fluka) at room temperature for 40 minutes. After three washes of 10 minutes at room temperature in PBS-Tween with 1% Skim Milk (Fluka) solution, the blot was incubated with monoclonal antibodies for eight hours at room temperature, gently stirring on a rotary shaker. After three washes for 10 minutes at room temperature in FSB-twin with 0.1% Skim Milk (Fluka), the blot was incubated for 2 hours with gentle shaking in FSB-twin with 0.1% solution of Skim Milk (Fluka), containing secondary antibodies conjugated to horseradish peroxidase. After washing the blot in FSB-Tween with a 0.1% Skim Milk solution (Fluka), the blot was placed in a substrate solution (3,3-diaminobenzidine (Sigma, USA); 0.003% H ₂ O) for 5 minutes. The reaction was stopped by washing the blot with water. The developed blot was dried and scanned. Protein expression was evaluated by Western blotting of the transformed cell lysate. To prepare the cell lysate, the same number of cells obtained after transformation with plasmids pCIGF-1 and pCIGF-lsynt was used (50,000 ng of plasmid were used for transformation). Cells were pelleted from the culture medium by centrifugation at 800 g and resuspended in lysis buffer. The same amount of cell lysate was applied to electrophoresis.

The results of Western blotting are presented in the drawing (see materials explaining the essence of the invention, figure 3). Using densitometric analysis, it was found that the brightness of the band corresponding to the expression of the synthetic gene FGsintIFR-1 is 47 times higher than the brightness of the band formed upon expression of the native gene IFR-1.

The following examples are given for the purpose of illustrating various embodiments of the invention and are not intended to limit the present invention in any way.

Example 1

The expression plasmids pCIGF-1 and pCIGF-lsynt were used to transform 293T eukaryotic cells. After transformation, the cells were incubated in 3 ml of culture medium for 48 hours. An enzyme-linked immunosorbent assay was performed on the culture medium of 293T cells transformed with these plasmids and expressing the native and synthetic IGF-1 genes. Using the enzyme-linked immunosorbent assay in the cell culture medium, antigen - protein IGF-1 was detected. The concentration of this protein corresponded to the expression level of the native and synthetic IGF-1 genes. In the case of the plasmid pCIGF-lsynt carrying FGintyntr-1, the maximum protein concentration was observed. The results of an enzyme-linked immunosorbent assay are presented in the drawing (see materials explaining the essence of the invention, figure 4).

The intensity of expression of the IGF-1 protein when using two variants of the gene can be roughly estimated as follows: IGF-1 - 1, FGsintIFR-1 - 100. Thus, FGsintIFR-1 can be effectively used both for gene therapy and for the production of IGF-1 as part of a eukaryotic producer.

Example 2

Experimental animals are rats.

Rats are a well-researched model for experimental studies of injuries. Each of 30 rats received 2 full-layer wounds under anesthesia and with anesthesia. Each wound site was round in shape with an approximate diameter of 2 cm, with a distance between the sites of approximately 5 cm, and was located in the upper part of the sides and on the back. Immediately after the induction of a wound in 10 animals, it was stabbed with a solution of a plasmid carrying the FGintyntr-1 gene. The other 10 animals received an injection of a plasmid carrying the native, more weakly expressed gene. The remaining 10 wounded animals were a control group. In rats treated with the pCIGF-lsynt and pCIGF-1 constructs, there was evidence that the IGF-1 mRNA was in the skin near the transfection sites, but not in the control tissues. In the case of using the plasmid construct pCIGF-lsynt, a statistically significant increase in the amount of mRNA by 14 times was observed.

The concentration of the protein IGF-1 was determined using ELISA in skin biopsies obtained from areas surrounding the wound.

In all skin biopsies taken from animals that were injected with pCIGF-lsynt and pCIGF-1 constructs, IGF-1 protein concentrations were determined along the wound perimeter as compared to biopsies taken from animals that were injected with empty plasmids or saline (p <0.01 ) The concentration of IGF-1 protein was statistically significantly higher by 12 times using the plasmid construct pCIGF-lsynt. There were no differences between the groups according to the concentration of IGF-1 protein in serum, liver, spleen or kidneys. In dynamics, the state of the bottom and edges of the wound was studied in all groups of animals using a semi-quantitative scale. To assess the effectiveness of the regenerative activity of the drugs used, the epithelization rate was measured. For this purpose, before the start of treatment, the area of the wound was precisely determined. To do this, a sterile sheet of plastic film was applied to the wound and the contours of the skin defect were transferred to it. Then, the resulting image of the wound was superimposed on a sheet of graph paper, after which the number of square centimeters and millimeters enclosed within the boundaries of the contour was counted. Repeated measurement of the wound area was carried out every 10-15 days of local and general therapy. The percentage reduction in the area of the wound during treatment was determined by the formula of L.N. Popova (1942) (Theory and practice of local treatment of purulent wounds. / E.P. Bezuglaya, S.G. Belov, V.G. Gunko et al. / Pod Edited by B.M. Datsenko. - K .: Zdorovya, 1995 .-- 384 p.). In addition, with each dressing, the maximum size of the ulcer was measured and photographed using a digital camera (Canon 350D). The results are presented in tables (see materials explaining the invention, FIG. 5 and FIG. 6).

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Claims (5)

1. The nucleotide sequence of the synthetic gene of insulin-like growth factor-1 (IGF-1) person, presented in the sequence listing SEQ ID NO: 1. 2. Recombinant plasmid DNA containing the nucleotide sequence of a synthetic gene of human insulin-like growth factor-1 (IGF-1) according to claim 1. 3. The eukaryotic cell containing the recombinant plasmid DNA according to claim 2 is a producer of human insulin-like growth factor-1 (IGF-1). 4. The design for gene therapy, containing the cytomegalovirus promoter, the nucleotide sequence of the synthetic gene of human insulin-like growth factor-1 (IGF-1) according to claim 1, the polyadenylation site, the resistance gene for ampicillin, the total size of 3662 nucleotides. 5. A pharmaceutical composition for gene therapy having a regenerative and wound healing effect, characterized in that it contains the construct according to claim 4 and a pharmaceutically acceptable carrier.

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