RU2700649C2 - Genetic construct based on non-viral vector plasmid including p4na2 gene cdna for reducing manifestations of human body conditions associated with reduced expression of p4na2 gene and/or reducing amount of prolyl 4-hydroxy acid alpha 2 protein, method of producing and use - Google Patents

Abstract

FIELD: biotechnology; medicine.

SUBSTANCE: invention relates to molecular biology, biotechnology, genetic engineering and medicine, and can be used for relief of manifestations of human body conditions associated with reduced expression of P4NA2 gene and/or reduced amount of protein prolyl 4-hydroxy acid alpha 2. Disclosed is a genetic construct based on a non-viral DNA vector comprising a P4NA2 gene cDNA selected from genetic constructs, each of which represents a genetic construct based on a DNA vector comprising a cDNA of the P4NA2 gene, with a coding protein sequence of prolyl 4-hydroxy-oxidase alpha 2, with 5' deletions and 3'-non-translated regions, specifically obtained based on the native non-modified cDNA region of the P4NA2 gene SEQ ID No: 1 or modified cDNA of the P4NA2 gene, wherein the modified P4NA2 gene cDNA used is SEQ ID No: 2, or SEQ ID No: 3, or SEQ ID No: 4, or SEQ ID No: 5, or SEQ ID No: 6, or SEQ ID No: 7, or a combination of these genetic constructs, each of which also contains regulatory elements providing a high level of expression of the P4NA2 gene in eukaryotic cells. Method of producing a genetic construct and use thereof are also provided.

EFFECT: invention allows increasing the amount of prolyl 4-hydroxylase alpha 2 protein in eukaryotic cells.

6 cl, 28 dwg, 20 ex

Description

FIELD OF THE INVENTION

The invention relates to molecular biology, biotechnology, genetic engineering and medicine, and can be used to correct the pathological conditions of cells of various organs and tissues, as well as human organs and tissues proper, associated with a decrease in the expression level of the P4NA2 gene and / or a decrease in the amount of protein shed 4- hydroxylase alpha 2, namely, for therapeutic purposes.

Prior level. Collagen is a fibrillar protein with a molecular weight of about 300 kDa, which is the most common protein in the human body. Collagen is the basis of connective tissue, the structural basis of the skin, cartilage, synovial fluid of the joints, bronchi, lung tissue, vascular wall, intervertebral discs, intestinal and stomach walls. In collagen, which is a glycoprotein, one third of the amino acid residues falls on glycine and another third on proline and hydroxyproline, which is a stabilizer and protector of the protein molecule from the action of proteases. Collagen provides the structural basis of tissue, gives it the necessary mechanical properties.

Collagen is synthesized in fibroblasts in the form of a high molecular weight precursor - procollagen. Collagen polypeptide chains enter the lumen of the endoplasmic reticulum in the form of propeptides with a signal sequence. Next, hydroxylation of the proline and lysine residues occurs, which is necessary for the correct folding of the newly synthesized procollagen chains.

The formation of hydroxyprolyl and hydroxylisyl is catalyzed by iron-containing enzymes - prolyl hydroxylase and lysyl hydroxylase, which are in microsomal fractions of many tissues. These enzymes are peptidyl hydroxylases, since hydroxylation occurs only after the inclusion of proline or lysine in the polypeptide chain.

Prolyl 4-hydroxylase is an oxidoreductase class enzyme that catalyzes hydroxylation at the 4 position of proline residues in synthesized procollagen chains. The enzyme requires the activity of Fe2 +, ascorbate and α-ketoglutarate for activity. The vertebrate enzyme isolated from all the studied sources has the structure of an alpha2beta2 tetramer; as a result of cloning and expression of the alpha subunit of C. elegans enzyme, it was found that the prolyl 4-hydroxylase of the latter is an alpha-beta dimer. The α-subunit 2 contains catalytic and peptide-substrate binding domains. This subunit is inactive in the absence of a β subunit. It is believed that there are no fundamental differences in the tissue distribution of both alpha subunits. The enzyme is an oxygenase with a mixed function and is active with the participation of molecular oxygen.

In vitro expression of active recombinant prolyl 4-hydroxylase from its subunits was successfully accomplished by transducing insect cells Spodoptera frugiperda and Trichoplusia ni (Vuori, K., et al. (1992) Proc Natl Acad Sci USA 89, 7467-7470) with recombinant baculoviruses or cotransfection of expression vectors in mammalian cell lines COS-1 (John, DC and Bulleid, NJ (1996) Biochem J 317 (Pt 3), 659-665) and HEK293 (Wagner, K, et al. (2000) Biochem J352 Pt 3, 907-911), in the yeast Pichia pastoris (Vuorela, A., et al. (1997) Embo J16, 6702-6712) and Saccharomyces cerevisiae (Toman, PD et al. (2000) J Biol Chem 275, 23303- 23309).

Expression systems for the production of recombinant collagens can also be E. coli cells, transgenic animals and plants. However, almost all of the above expression systems have their drawbacks. For example, when an enzyme is obtained in E. coli cells, there is no post-translational modification of the protein, and when obtained in yeast cells, the recombinant protein does not have prolyl-4-hydroxylase activity. The expression system in mammalian cells has insufficient production. As for transgenic animals (silkworm or mice) or plants (tobacco), collagen is produced in them, the molecules of which are connected by cross-linking, as a result of which the purification of such a product is very difficult. Therefore, the creation of an effective expression system for producing collagen in high yield is very important.

The known P4NA2 gene shed human 4-hydroxylase encoding the catalytic α (II) subunit of the main isoenzyme (https://www.ncbi.nlm.nih.gov/gene/8974). The P4NA2 gene is localized at position 5q31.1, and has a length of 37716 bp. and consists of 18 exons. The gene product catalyzes the formation of 4-hydroxyproline, which is necessary for the correct folding of the synthesized procollagen chains.

It has been shown that high degree myopia, being one of the main reasons for the development of blindness, can be caused by mutations in the P4NA2 gene encoding the subunit of alpha-2 shed human 4-hydroxylases (Guo H, Tong P, Liu Y, Xia L, Wang T, Tian Q, Li Y, Hu Y, Zheng Y, Jin X, Li Y, Xiong W, Tang B, Feng Y, Li J, Pan Q, Hu Z, Xia K. Mutations of P4NA2 encoding prolyl 4-hydroxylase 2 are associated with nonsyndromic high myopia. Genet Med. 2015 17 (4): 300-6.) As a result of a genome-wide screening of 186 people, including those with familial myopathy, it was revealed that the c.871G> A mutation leads to the above phenotype. A mechanism for the development of pathology was proposed, according to which the degradation of messenger RNA carrying the mutation occurs.

WO 2014170460 (A2) describes a method for producing collagen from sea sponges (Chondrosia reniformis) in Pichia pastoris yeast. Three vector designs were created:

- with cDNA encoding the alpha subunit of prolyl 4-hydroxylase (pPinkHC and pPinkLC vectors),

with cDNA encoding the beta subunit of prolyl 4-hydroxylase (pPIC6B \ PDI vector),

- with cDNA encoding non-fibrillar collagen protein C. Reniformis (pPICZB \ ColCH).

The application describes a method that involves the joint transformation of a yeast strain with two different vectors containing: the first is the coding sequence of the alpha subunit, the second is the coding sequence of the beta subunit of the prolyl 4-hydroxylase enzyme, followed by transformation using a vector containing one sequence, coding collagen of a sea sponge.

The disadvantage of this approach is that the yeast cell expression system, which according to the literature does not have significant prolyl-4-hydroxylase activity, was selected for the expression of the genes of prolyl 4-hydroxylase subunits of the sea sponge. This patent application does not consider the possibility of expression in human cells as a gene-therapeutic agent, taking into account the individual characteristics of the patient, in connection with which a group of variations for this agent may be needed.

For the prototype, the authors decided on patent US 7759090 (B2), which describes the expression system for producing collagen. Stably transfected insect cell lines containing cDNA coding for human α-and β-subunits of prolyl 4-hydroxylase in Trichoplusia ni and Drosophila melanogaster S2 cells are shown. Additionally, the involvement of prolyl 4-hydroxylase in the assembly of three alpha chains with the formation of trimeric type minicollagen XXI, which contains the intact C-terminal non-collagen domain (NC1) and collagen domain (COL1) in the Drosophila system, was characterized. Minicollagen XXI coexpressed with prolyl 4-hydroxylase contained sufficient hydroxyproline to form heat-resistant pepsin-resistant triple helices.

One embodiment of the invention describes insect cells transfected with a gene encoding prolyl 4-hydroxylase. This gene consists of the α subunit and / or β subunit of prolyl 4-hydroxylase. Insect cells are additionally transfected with a gene encoding the collagen (COL1) domain and the C1-terminal domain (NC1) of type XXI collagen.

Genes encoding the α subunit and β subunit of human prolyl 4-hydroxylase were cloned together in the plZ / V5-His expression vector under the control of the OplE2 promoter. In the Drosophila inducible expression (DES®) system (Invitrogen), genes encoding the α subunit and β-subunit of prolyl 4-hydroxylase were cloned separately in the pMT / 5-HisAA expression vector under the control of the metallothionein promoter, which is induced by the addition of copper or cadmium ions.

It was shown that P4Hα and P4Hβ subunits are able to assemble into the active tetramer α2β2. The obtained prolyl 4-hydroxylase provides the synthesis of correctly folded collagen. A comparison of the expression levels of P4Hα and P4Hβ in the lysates of Trichoplusia ni cells showed that the expression level of the P4Hα protein is approximately 5 times lower than P4Hβ. For P4H expression in the inducible Drosophila expression system, Drosophila S2 cells were co-transfected with pMT / P4Hα, pMT / P4Hβ and pCoHygro in a 10: 10: 1 ratio using Effectene transfection reagent (Qiagen). Collagen domains were introduced into cells by transformation with the pMT / BiP-mC21 vector. Thus, recombinant collagen was obtained using the expression system of three genes in Drosophila melanogaster cells.

The disadvantage of this approach is that for the expression of human prolyl 4-hydroxylase subunit genes, an insect cell expression system was selected which, according to published data, has low prolyl 4-hydroxylase activity. In addition, there is evidence of insufficient efficiency and correctness of post-translational modifications in insect cells compared to human cells. Also, the possibility of expression in human cells as a gene-therapeutic agent is not considered, taking into account the individual characteristics of the patient, in connection with which a group of variations for this agent may be needed.

Disclosure of invention

The objective of the invention is the creation of a genetic construct based on a non-viral vector plasmid, including cDNA of the P4NA2 gene, for stopping the manifestations of human body conditions associated with a decrease in the expression level of the P4HA2 gene and / or a decrease in the amount of protein shed by 4-hydroxylase alpha 2 selected from the group of genetic constructs with cDNA of the P4NA2 gene, using which, taking into account the individual characteristics of the patient, there is an increase in the expression level of the P4NA2 gene and / or an increase in the amount of protein shed 4-hydroxylase alpha 2 in the cells of organs and tissues and / or organs and tissues of the body.

This problem is solved due to the fact that a genetic construct based on a non-viral DNA vector including cDNA of the P4NA2 gene was created to stop the manifestations of human body conditions associated with a decrease in the expression of the P4HA2 gene and / or a decrease in the amount of protein shed by 4-hydroxylase alpha 2, selected from the group of genetic constructs, each of which represents a genetic construct based on a DNA vector including cDNA of the P4NA2 gene, with the coding sequence of the protein, shed 4-hydroxylase alpha 2, with 5'and 3'-untranslated regions, namely, obtained on the basis of a portion of a native unmodified cDNA of the P4NA2 gene SEQ ID No: 1, or a modified cDNA of the P4NA2 gene, while SEQ ID No: 2 is used as a modified cDNA of the P4NA2 gene, or SEQ ID No: 3, or SEQ ID No: 4, or SEQ ID No: 5, or SEQ ID No: 6, or SEQ ID No: 7 or a combination of these genetic constructs, each of which also contains regulatory elements that provide a high level expression of the P4NA2 gene in eukaryotic cells, namely, in cells of human organs and tissues, and capable of increasing the amount of protein shed by 4-hydroxylase alpha 2 in the cells of organs and tissues and / or human organs and tissues, namely, hematopoietic cells, or mesenchymal stem cells, or chondroblasts, or myocytes, or myoblasts, or fibroblasts, or osteoblasts, or keratocytes, or epithelial cells, or corneal cells, or endothelial cells, or epithelial cells, in combination with or without a transport molecule when transfected with these genetic constructs, cells of human organs and tissues, and / or in organs and tissues x person, namely, in connective tissue structures, or skin, or joints, or cartilage, or bone, or muscle, or tendons, or ligaments, or blood vessels, or the wall of arteries, or the cornea of the eye, or sclera , or the placenta, or dentine, or the stroma of internal organs in combination with or without a transport molecule when these genetic constructs are introduced into human organs and tissues. Moreover, each genetic construct selected from the group of genetic constructs with a modified cDNA of the P4NA2 gene contains a nucleotide sequence that includes a protein coding region of the cDNA of the P4HA2 gene, which carries modifications that do not affect the protein structure; shed 4-hydroxylase alpha 2 or affect the protein structure shed 4-hydroxylase alpha 2 and affecting the amino acid sequence encoded by this sequence, namely: nucleotide substitutions leading to amino acid substitutions or termination amino acid chain, and / or deletions, or combinations of these modifications. Moreover, liposomes, or dendrimers of the 5th and higher generations, or amphiphilic block copolymers, or a polyethyleneimine-based reagent are used as a transport molecule.

A method of obtaining a genetic construct is to obtain each genetic construct selected from the group of genetic constructs, whereby cDNA of the P4NA2 gene is obtained, then cDNA is placed in a vector plasmid capable of providing a high level of expression of this cDNA in cells of various human organs and tissues, and the necessary the amount of genetic construct, then combine the genetic construct with a transport molecule to transfect the resulting genetic construct of organ cells and tissues and / or introducing the obtained genetic construct into human organs and tissues, using cDNA of the P4NA2 gene with the coding sequence of the protein shed 4-hydroxylase alpha 2, with divisions of 5 'and 3'-untranslated regions, namely, obtained on the basis of the native unmodified cDNA of the P4NA2 gene SEQ ID No: 1, or a modified cDNA of the P4HA2 gene, while either SEQ ID No: 2, or SEQ ID No: 3, or SEQ ID No: 4, or SEQ ID as a modified cDNA of the P4HA2 gene No: 5, or SEQ ID No: 6, or SEQ ID No: 7, or a combination of these genetic constructs.

A method of using a genetic construct consists in transfecting a genetic construct or several genetic constructs selected from the group of created genetic constructs of cells of organs and tissues of a patient and / or introducing into the organs and tissues of a patient autologous cells of a patient transfected with a genetic construct or several genetic constructs selected from the group created genetic constructs, and / or in the introduction into the organs and tissues of a patient of a genetic construct or several their genetic constructs selected from the group created by the genetic constructs or methods of combination indicated. At the same time, the choice of the genetic construct from the created genetic constructs is made individually for each patient, after a preliminary determination of the most effective genetic construct for this patient has been made.

List of figures

In FIG. one

The nucleotide sequence of the unmodified cDNA of the P4NA2 gene is presented, the sequence of which is homologous to that given in the GenBank database under the number NM_004199 P4NA2 SEQ ID No: 1.

In FIG. 2

The nucleotide sequence of the P4NA2 cDNA thus modified SEQ ID No: 2 contains 2 nucleotide substitutions G → C at positions 1089, 1119; 1 nucleotide substitution T → G at position 1239, 1 nucleotide substitution A → G at position 1290; which do not lead to changes in the amino acid sequence of the protein encoded by the sequence of the P4HA2 gene and has a primary structure shown in FIG. 2:

In FIG. 3

The nucleotide sequence of the P4NA2 cDNA thus modified SEQ ID No: 3 contains 3 nucleotide substitutions G → C at positions 1089, 1119, 1347; 4 nucleotide substitutions T → G at positions 1239, 1449, 1476, 1665, 2 nucleotide substitutions A → G at positions 1290, 1653; which do not lead to changes in the amino acid sequence of the protein encoded by the sequence of the P4HA2 gene and has a primary structure shown in FIG. 3:

In FIG. four

The nucleotide sequence of the P4NA2 cDNA thus modified SEQ ID No: 4 contains 4 nucleotide substitutions G → C at positions 1089, 1119, 1347, 1905; 6 nucleotide substitutions T → G at positions 1239, 1449, 1476, 1665, 1764, 1914; 6 nucleotide substitutions A → G at positions 1290, 1653, 1755, 1881, 2058, 2127; which do not lead to changes in the amino acid sequence of the protein encoded by the sequence of the P4HA2 gene and has a primary structure shown in FIG. four:

In FIG. 5

The nucleotide sequence of the P4NA2 cDNA thus modified SEQ ID No: 5, length 1602 n.p. contains a deletion of 6 nucleotide pairs, as well as a substitution of 8 amino acid residues, leading to changes in the amino acid sequence of the protein shed by 4-hydroxylase alpha 2 and has the primary structure shown in FIG. 5.

In FIG. 6

The thus modified nucleotide sequence of the P4NA2 cDNA SEQ ID No: 6, length 1602 n.p. contains 3 nucleotide substitutions G → C at positions 1089, 1119, 1347; 2 nucleotide substitutions T → G at positions 1239, 1449, 1 nucleotide substitution A → G at position 1290; a deletion of 6 nucleotide pairs, as well as a replacement of 8 amino acid residues, leading to changes in the amino acid sequence of the protein shed by 4-hydroxylase alpha 2 and has the primary structure shown in FIG. 6:

In FIG. 7

The nucleotide sequence of the P4NA2 cDNA thus modified SEQ ID No: 7, length 1602 n.p. contains 3 nucleotide substitutions G → C at positions 1089, 1119, 1347; 5 nucleotide substitutions T → G at positions 1239, 1449, 1476, 1665, 1764; 5 nucleotide substitutions A → G at positions 1290, 1653, 1755, 2058, 2127; a deletion of 6 nucleotide pairs, as well as a replacement of 8 amino acid residues, leading to changes in the amino acid sequence of the protein shed by 4-hydroxylase alpha 2 and has the primary structure shown in FIG. 7:

In FIG. 8

For the subsequent correct determination of the gene therapeutic effect after transfection of fibroblasts with a genetic construct with cDNA of the P4NA2 - VTvaf17 - P4HA2 gene SEQ ID No: 1, an analysis of the endogenous expression of the P4HA2 gene in a primary fibroblast culture was performed. The figure shows the graphs of the accumulation of products of polymerase chain reaction (PCR), corresponding to:

1 - cDNA of the P4HA2 gene, fibroblasts with reduced expression of the P4HA2 gene

2 - cDNA of the P4HA2 gene, fibroblasts with normal expression of the P4HA2 gene

3 - cDNA of the B2M gene, fibroblasts with reduced expression of the P4HA2 gene

4 - cDNA of the B2M gene, fibroblasts with normal expression of the P4HA2 gene

The B2M gene (Beta-2-microglobulin) shown in the GenBank database under the number NM 004048.2 was used as the reference gene.

In FIG. 9

In order to confirm the increase in the expression of the P4HA2 gene in a cell culture of fibroblasts with reduced expression of the P4HA2 gene during transfection of these cells with the VTvaf17 genetic construct - P4HA2 SEQ ID No: 1 from the cDNA of the P4HA2 gene, the plots of accumulation of PCR products corresponding to:

1 - cDNA of the P4NA2 gene in fibroblasts with normal expression of the P4NA2 gene,

2 - cDNA of the P4NA2 gene in fibroblasts with reduced expression of the P4NA2 gene prior to transfection with the cDNA construct of the P4NA2 gene

3 - cDNA of the P4NA2 gene in fibroblasts with reduced expression of the P4NA2 gene after transfection with the cDNA construct of the P4NA2 gene

4 - cDNA of the P4NA2 gene in fibroblasts with reduced expression of the P4NA2 gene after transfection with a vector without cDNA of the P4NA2 gene

5 - cDNA of the B2M gene in fibroblasts with normal expression of the P4NA2 gene,

6 - cDNA of the B2M gene in fibroblasts with reduced expression of the P4NA2 gene before transfection with the cDNA construct of the P4NA2 gene

7 - cDNA of the B2M gene in fibroblasts with reduced expression of the P4NA2 gene after transfection with the cDNA construct of the P4NA2 gene

8 - cDNA of the B2M gene in fibroblasts with reduced expression of the P4NA2 gene after transfection with a vector without cDNA of the P4NA2 gene

From the graphs it follows that in the case of transfection with a vector without inserting a cDNA of the P4NA2 gene, the cDNA level of the P4NA2 gene in fibroblasts did not change, and in the case of transfection with the VTvaf17 construct - P4NA2 SEQ ID No: 1 with P4NA2 cDNA - the level of fibroblast cDNA with decreased P4NA2 gene expression significantly increased .

In FIG. 10

In order to confirm the increase in the expression of the P4NA2 gene in cell culture of fibroblasts with reduced expression of the P4HA2 gene during transfection of these cells with the genetic construct VTvaf17-P4HA2 SEQ ID No: 2 with cDNA of the P4NA2 gene, plots of accumulation of PCR products are presented that correspond to:

1 - cDNA of the P4HA2 gene in fibroblasts with normal expression of the P4HA2 gene,

2 - cDNA of the P4HA2 gene in fibroblasts with reduced expression of the P4HA2 gene before transfection with the cDNA construct of the P4HA2 gene

3 - cDNA of the P4HA2 gene in fibroblasts with reduced expression of the P4HA2 gene after transfection with the cDNA construct of the P4HA2 gene

4 - cDNA of the P4HA2 gene in fibroblasts with reduced expression of the P4HA2 gene after transfection with a vector without cDNA of the P4HA2 gene

5 - cDNA of the B2M gene in fibroblasts with normal expression of the P4HA2 gene,

6 - cDNA of the B2M gene in fibroblasts with reduced expression of the P4HA2 gene before transfection with the cDNA construct of the P4HA2 gene

7 - cDNA of the B2M gene in fibroblasts with reduced expression of the P4HA2 gene after transfection with the cDNA construct of the P4HA2 gene

8 - cDNA of the B2M gene in fibroblasts with reduced expression of the P4HA2 gene after transfection with a vector without cDNA of the P4HA2 gene

From the graphs it follows that in the case of transfection with a vector without inserting cDNA of the P4HA2 gene, the cDNA level of the P4HA2 gene in fibroblasts did not change, and in the case of transfection with the VTvaf17-P4HA2 SEQ ID No: 2 construct with P4HA2 cDNA, the level of fibroblast cDNA with significantly reduced P4HA2 gene expression .

In FIG. eleven

In order to confirm the increase in the expression of the P4HA2 gene in a cell culture of fibroblasts with reduced expression of the P4HA2 gene during transfection of these cells with the VTvaf17-P4HA2 genetic construct SEQ ID No: 3 with cDNA of the P4NA2 gene, plots of accumulation of PCR products corresponding to:

1 - cDNA of the P4NA2 gene in fibroblasts with normal expression of the P4NA2 gene,

2 - cDNA of the P4NA2 gene in fibroblasts with reduced expression of the P4NA2 gene prior to transfection with the cDNA construct of the P4NA2 gene

3 - cDNA of the P4NA2 gene in fibroblasts with reduced expression of the P4NA2 gene after transfection with the cDNA construct of the P4NA2 gene

4 - cDNA of the P4NA2 gene in fibroblasts with reduced expression of the P4NA2 gene after transfection with a vector without cDNA of the P4NA2 gene

5 - cDNA of the B2M gene in fibroblasts with normal expression of the P4NA2 gene,

6 - cDNA of the B2M gene in fibroblasts with reduced expression of the P4NA2 gene before transfection with the cDNA construct of the P4NA2 gene

7 - cDNA of the B2M gene in fibroblasts with reduced expression of the P4NA2 gene after transfection with the cDNA construct of the P4NA2 gene

8 - cDNA of the B2M gene in fibroblasts with reduced expression of the P4NA2 gene after transfection with a vector without cDNA of the P4NA2 gene

From the graphs it follows that in the case of transfection with a vector without inserting cDNA of the P4NA2 gene, the cDNA level of the P4NA2 gene in fibroblasts did not change, and in the case of transfection with the VTvaf17-P4NA2 construct SEQ ID No: 3 with P4NA2 cDNA, the level of fibroblast cDNA with decreased P4NA2 gene expression significantly increased .

In FIG. 12

In order to confirm the increase in the expression of the P4NA2 gene in a cell culture of fibroblasts with reduced expression of the P4HA2 gene during transfection of these cells with the VTvaf17-P4NA2 genetic construct SEQ ID No: 4 with cDNA of the P4NA2 gene, plots of accumulation of PCR products corresponding to:

1 - cDNA of the P4NA2 gene in fibroblasts with normal expression of the P4NA2 gene,

2 - cDNA of the P4NA2 gene in fibroblasts with reduced expression of the P4NA2 gene prior to transfection with the cDNA construct of the P4NA2 gene

3 - cDNA of the P4NA2 gene in fibroblasts with reduced expression of the P4NA2 gene after transfection with the cDNA construct of the P4NA2 gene

4 - cDNA of the P4NA2 gene in fibroblasts with reduced expression of the P4NA2 gene after transfection with a vector without cDNA of the P4NA2 gene

5 - cDNA of the B2M gene in fibroblasts with normal expression of the P4NA2 gene,

6 - cDNA of the B2M gene in fibroblasts with reduced expression of the P4NA2 gene before transfection with the cDNA construct of the P4NA2 gene

7 - cDNA of the B2M gene in fibroblasts with reduced expression of the P4NA2 gene after transfection with the cDNA construct of the P4NA2 gene

8 - cDNA of the B2M gene in fibroblasts with reduced expression of the P4NA2 gene after transfection with a vector without cDNA of the P4NA2 gene

From the graphs it follows that in the case of transfection with a vector without inserting a cDNA of the P4NA2 gene, the cDNA level of the P4NA2 gene in fibroblasts did not change, and in the case of transfection with the VTvaf17-P4NA2 construct SEQ ID No: 4 with P4NA2 cDNA, the level of fibroblast cDNA with decreased P4NA2 gene expression significantly increased .

In FIG. 13

In order to confirm the increase in the expression of the P4NA2 gene in a cell culture of fibroblasts with reduced expression of the P4HA2 gene during transfection of these cells with the VTvaf17-P4NA2 genetic construct SEQ ID No: 5 with cDNA of the P4NA2 gene, the plots of the accumulation of PCR products corresponding to:

1 - cDNA of the P4NA2 gene in fibroblasts with normal expression of the P4NA2 gene,

2 - cDNA of the P4NA2 gene in fibroblasts with reduced expression of the P4NA2 gene prior to transfection with the cDNA construct of the P4NA2 gene

3 - cDNA of the P4NA2 gene in fibroblasts with reduced expression of the P4NA2 gene after transfection with the cDNA construct of the P4NA2 gene

4 - cDNA of the P4NA2 gene in fibroblasts with reduced expression of the P4NA2 gene after transfection with a vector without cDNA of the P4NA2 gene

5 - cDNA of the B2M gene in fibroblasts with normal expression of the P4NA2 gene,

6 - cDNA of the B2M gene in fibroblasts with reduced expression of the P4NA2 gene before transfection with the cDNA construct of the P4NA2 gene

7 - cDNA of the B2M gene in fibroblasts with reduced expression of the P4NA2 gene after transfection with the cDNA construct of the P4NA2 gene

8 - cDNA of the B2M gene in fibroblasts with reduced expression of the P4NA2 gene after transfection with a vector without cDNA of the P4NA2 gene

From the graphs it follows that in the case of transfection with a vector without inserting a cDNA of the P4NA2 gene, the cDNA level of the P4NA2 gene in fibroblasts did not change, and in the case of transfection with the VTvaf17-P4NA2 construct SEQ ID No: 5 with P4NA2 cDNA, the level of fibroblast cDNA with decreased P4NA2 gene expression significantly increased .

In FIG. fourteen

In order to confirm the increase in the expression of the P4NA2 gene in a cell culture of fibroblasts with reduced expression of the P4HA2 gene during transfection of these cells with the VTvaf17-P4HA2 genetic construct SEQ ID No: 6 with cDNA of the P4NA2 gene, plots of accumulation of PCR products corresponding to:

1 - cDNA of the P4NA2 gene in fibroblasts with normal expression of the P4NA2 gene,

2 - cDNA of the P4NA2 gene in fibroblasts with reduced expression of the P4NA2 gene prior to transfection with the cDNA construct of the P4NA2 gene

3 - cDNA of the P4NA2 gene in fibroblasts with reduced expression of the P4NA2 gene after transfection with the cDNA construct of the P4NA2 gene

4 - cDNA of the P4NA2 gene in fibroblasts with reduced expression of the P4NA2 gene after transfection with a vector without cDNA of the P4NA2 gene

5 - cDNA of the B2M gene in fibroblasts with normal expression of the P4NA2 gene,

6 - cDNA of the B2M gene in fibroblasts with reduced expression of the P4NA2 gene before transfection with the cDNA construct of the P4NA2 gene

7 - cDNA of the B2M gene in fibroblasts with reduced expression of the P4NA2 gene after transfection with the cDNA construct of the P4NA2 gene

8 - cDNA of the B2M gene in fibroblasts with reduced expression of the P4NA2 gene after transfection with a vector without cDNA of the P4NA2 gene

From the graphs it follows that in the case of transfection with a vector without inserting a cDNA of the P4NA2 gene, the cDNA level of the P4NA2 gene in fibroblasts did not change, and in the case of transfection with the VTvaf17-P4HA2 SEQ ID No: 6 construct with P4NA2 cDNA, the level of fibroblast cDNA with significantly reduced P4HA2 gene expression .

In FIG. fifteen

In order to confirm the increase in the expression of the P4HA2 gene in a cell culture of fibroblasts with reduced expression of the P4HA2 gene during transfection of these cells with the VTvaf17-P4HA2 SEQ ID No: 7 genetic construct from the cDNA of the P4HA2 gene, plots of accumulation of PCR products corresponding to:

1 - cDNA of the P4HA2 gene in fibroblasts with normal expression of the P4HA2 gene,

2 - cDNA of the P4HA2 gene in fibroblasts with reduced expression of the P4HA2 gene before transfection with the cDNA construct of the P4HA2 gene

3 - cDNA of the P4HA2 gene in fibroblasts with reduced expression of the P4HA2 gene after transfection with the cDNA construct of the P4HA2 gene

4 - cDNA of the P4HA2 gene in fibroblasts with reduced expression of the P4HA2 gene after transfection with a vector without cDNA of the P4HA2 gene

5 - cDNA of the B2M gene in fibroblasts with normal expression of the P4HA2 gene,

6 - cDNA of the B2M gene in fibroblasts with reduced expression of the P4HA2 gene before transfection with the cDNA construct of the P4HA2 gene

7 - cDNA of the B2M gene in fibroblasts with reduced expression of the P4HA2 gene after transfection with the cDNA construct of the P4HA2 gene

8 - cDNA of the B2M gene in fibroblasts with reduced expression of the P4HA2 gene after transfection with a vector without cDNA of the P4HA2 gene

From the graphs it follows that in the case of transfection with a vector without inserting a cDNA of the P4HA2 gene, the cDNA level of the P4HA2 gene in fibroblasts did not change, and in the case of transfection with the VTvaf17-P4HA2 SEQ ID No: 7 construct with P4NA2 cDNA, the level of fibroblast cDNA with significantly reduced P4HA2 gene expression .

In FIG. 16

In order to confirm the increase in the amount of protein, 4-hydroxylase alpha 2 was shed in a cell culture of fibroblasts with normal expression of the P4HA2 gene during transfection of these cells with the genetic construct of the P4HA2 cDNA gene, a graph of the changes in the amount of protein shed of 4-hydroxylase alpha 2 of non-transfected fibroblasts (culture A) transfected with pCDNA 3.1 (+) vector not containing P4HA2 cDNA (culture B) and transfected with pCDNA 3.1-P4HA2 genetic construct SEQ ID No: 2 (culture C). It follows from the graph that upon transfection of fibroblasts with a genetic construct with cDNA of the P4HA2 gene, an increase in the amount of protein shed by 4-hydroxylase alpha 2 in the cell lysate occurs.

In FIG. 17

In order to confirm the increase in the amount of protein, 4-hydroxylase alpha 2 was shed in human skin when a cell culture of fibroblasts transfected with the genetic construct containing the P4HA2 gene cDNA was transfected into the skin, a diagram of the changes in the amount of protein shed of 4-hydroxylase alpha 2 in the skin of patients was presented. In this case, patients received three culture variants of autologous fibroblasts — non-transfected (zone A), transfected with the VTvaf17 vector (zone B) and transfected with the genetic construct VTvaf17-P4HA2 SEQ ID No: 7 (zone C) - into the skin of the forearm. The quantitative level of the protein was also analyzed by shedding 4-hydroxylase alpha 2 in intact skin. An increase in the amount of protein shed by 4-hydroxylase alpha 2 in the patient’s skin in the area of the introduction of fibroblasts transfected with the genetic construct with cDNA of the P4HA2 gene (C) was shown.

In FIG. eighteen

In order to confirm the increase in the amount of protein, 4-hydroxylase alpha 2 was shed to various individual levels in cell cultures of fibroblasts of patients upon transfection of these cells with genetic constructs with modified and native cDNA of the P4HA2 gene, depending on the presence and type of a particular modification of the c4DNA of the P4HA2 gene, the analysis of changes in the quantitative level of the protein shed by 4-hydroxylase alpha 2 in cultures of human skin fibroblasts is presented, depending on the presence and type of modifications in the cDNA of the P4HA2 gene, we use oh for transfection of fibroblasts.

Fibroblast cultures of 50 patients were divided into 8 parts each from (A) to (Z); the first parts (A) of patient cell cultures were transfected with the pCMV6-XL5 P4HA2 genetic construct SEQ ID No: 1, the parts (B) were transfected with the pCMV6-XL5 genetic construct P4HA2 SEQ ID No: 2, parts (C) were transfected with the pCMV6-XL5 P4HA2 SEQ genetic construct ID No: 3, part (D) was transfected with the genetic construct pCMV6-XL5 P4HA2 SEQ ID No: 4, part (E) was transfected with the genetic construct pCMV6-XL5 P4HA2 SEQ ID No: 5, part (F) was transfected with the genetic construct pCMV6-XL5 P4HA2 SEQ ID No: 6, part (G) was transfected with the genetic construct pCMV6-XL5 P4NA2 SEQ ID No: 7, part (H) was transfected were infected with the vector plasmid pCMV6-XL5, which did not contain the cDNA of the P4NA2 gene.

Based on the results of the analysis of the quantitative level of the protein, 4-hydroxylase alpha 2 shedding selected indicators for each part of the cell culture from each patient, showing the maximum amount of protein shedding 4-hydroxylase alpha 2 and combined them into groups based on the following criterion:

In group 1, the maximum amount of protein shed by 4-hydroxylase alpha 2 was observed during transfection of pCMV6-XL5 P4NA2 SEQ ID No: 1,

in group 2, the maximum amount of protein shed by 4-hydroxylase alpha 2 was observed during transfection of pCMV6-XL5 P4NA2 SEQ ID No: 2,

in group 3, the maximum amount of protein shed by 4-hydroxylase alpha 2 was observed during transfection of pCMV6-XL5 P4NA2 SEQ ID No: 3,

in group 4, the maximum amount of protein shed by 4-hydroxylase alpha 2 was observed during transfection of pCMV6-XL5 P4NA2 SEQ ID No: 4,

in group 5, the maximum amount of protein shed by 4-hydroxylase alpha 2 was observed during transfection of pCMV6-XL5 P4NA2 SEQ ID No: 5,

in group 6, the maximum amount of protein shed by 4-hydroxylase alpha 2 was observed during transfection of pCMV6-XL5 P4NA2 SEQ ID No: 6,

in group 7, the maximum amount of protein shed by 4-hydroxylase alpha 2 was observed upon transfection with pCMV6-XL5 P4NA2 SEQ ID No: 7.

In none of the cell cultures was it observed that the maximum amount of protein shed by 4-hydroxylase alpha 2 was present upon transfection with the pCMV6-XL5 vector without insertion of the cDNA of the P4NA2 gene.

Figure 18 shows, for each group of cell cultures, a table of indicators of protein concentration shedding 4-hydroxylase alpha 2 (averaged within the group if more than one cell culture is included in the group) for all genetic constructs participating in the experiment after transfection of these cell cultures with genetic constructs containing modified and native cDNA of the P4NA2 gene.

It follows from the figure that the maximum amount of protein was shed by 4-hydroxylase alpha 2 in the skin fibroblast cultures of various patients during their transfection with genetic constructs is related to the individual characteristics of the patients and depends on the presence and type of modifications in the P4NA2 gene cDNA integrated into the genetic constructs.

Each genetic construct from the group of genetic constructs is effective in some significant group of patients. Therefore, in order to select the most effective genetic construct from the group of genetic constructs for therapeutic purposes, a preliminary personalized study of the patient is necessary.

In FIG. 19

In order to confirm the increase in the expression of the P4NA2 gene in a cell culture of keratocytes and epithelial cells of the cornea of the eye during transfection of these cells with the pCMV6-Kan / Neo P4HA2 SEQ ID No: 2 construct with cDNA of the P4NA2 gene, plots of accumulation of PCR products corresponding to:

1 - cDNA of the P4HA2 gene, keratocytes prior to transfection with the cDNA construct of the P4HA2 gene

2 - cDNA of the P4HA2 gene, corneal epithelium before transfection with the cDNA construct of the P4HA2 gene

3 - cDNA of the P4HA2 gene, keratocytes after transfection with the cDNA construct of the P4HA2 gene

4 - cDNA of the P4HA2 gene, corneal epithelium after transfection with the c4DNA construct of the P4HA2 gene

5 - cDNA of the B2M gene, keratocytes before transfection with the c4DNA construct of the P4HA2 gene

6 - cDNA of the B2M gene, corneal epithelium prior to transfection with the c4DNA construct of the P4HA2 gene

7 - cDNA of the B2M gene, keratocytes after transfection with the c4DNA construct of the P4HA2 gene

8 - cDNA of the B2M gene, corneal epithelium after transfection with the cDNA construct of the P4HA2 gene

The B2M gene was used as a reference.

It follows from the figure that as a result of transfection with the pCMV6-Kan / Neo P4HA2 genetic construct SEQ ID No: 2, the level of specific cDNA of the P4HA2 gene in the culture of keratocytes and in the culture of the corneal epithelium increased many times.

In FIG. twenty

In order to confirm the increase in the expression of the P4HA2 gene in the primary culture of smooth muscle cells of the human bladder HBdSMC (ATCC PCS-420-012) upon transfection of these cells with the genetic construct GDTT1.8NAS1-P4HA2 SEQ ID No: 3 with cDNA of the P4NA2 gene, plots of accumulation of PCR products are shown corresponding to:

1 - cDNA of the P4NA2 gene prior to transfection with the genetic construct GDTT1.8NAS1-P4NA2 SEQ ID No: 3, carrying the region of the P4NA2 gene ;;

2 - cDNA of the P4NA2 gene after transfection with the genetic construct GDTT1.8NAS1-P4NA2 SEQ ID No: 3, carrying a portion of the P4NA2 gene;

3 - cDNA of the B2M gene before transfection with the genetic construct GDTT1.8NAS1-P4NA2 SEQ ID No: 3, carrying a portion of the P4NA2 gene;

4 - cDNA of the B2M gene after transfection with the genetic construct GDTT1.8NAS1-P4NA2 SEQ ID No: 3, carrying a portion of the P4NA2 gene;

The B2M gene was used as a reference.

It follows from the figure that as a result of transfection with the genetic construct GDTT1.8NAS1-P4NA2 SEQ ID No: 3, the level of specific cDNA of the P4NA2 gene in the human bladder smooth muscle cell culture of HBdSMC increased many times.

In FIG. 21

In order to confirm the increase in the expression of the P4NA2 gene in a cell culture of chondroblasts during transfection of these cells with the pCMV6-Kan / Neo P4NA2 genetic construct SEQ ID No: 4 with cDNA of the P4NA2 gene, the plots of accumulation of PCR products corresponding to:

1 - cDNA of the P4NA2 gene, before transfection with pCMV6-Kan / Neo P4NA2 SEQ ID No: 4

2 - cDNA of the P4NA2 gene, after transfection with pCMV6-Kan / Neo P4NA2 SEQ ID No: 4

3 - cDNA of the B2M gene, before transfection with pCMV6-Kan / Neo P4NA2 SEQ ID No: 4

4 - cDNA of the B2M gene, after transfection with pCMV6-Kan / Neo P4NA2 SEQ ID No: 4

The B2M gene was used as a reference.

It follows from the figure that as a result of transfection of chondroblasts with the pCMV6-Kan / Neo P4NA2 genetic construct SEQ ID No: 3, the level of specific c4NA of the P4NA2 gene increased many times.

In FIG. 22

In order to confirm the increase in the expression of the P4NA2 gene in the primary culture of endothelial cells from the human umbilical vein HUVEC (ATCC ^® CRL-1730 ™) upon transfection of these cells with the genetic construct GDTT1.8NAS3-P4HA2 SEQ ID No: 4 with the cDNA of the P4NA2 gene, plots of PCR accumulation are shown products matching:

1 - cDNA of the P4NA2 gene prior to transfection with the genetic construct GDTT1.8NAS3-P4HA2 SEQ ID No: 4, carrying the portion of the P4NA2 gene ;;

2 - cDNA of the P4NA2 gene after transfection with the genetic construct GDTT1.8NAS3-P4HA2 SEQ ID No: 4, carrying a portion of the P4NA2 gene;

3 - cDNA of the B2M gene prior to transfection with transfection with the genetic construct GDTT1.8NAS3-P4HA2 SEQ ID No: 4, carrying a portion of the P4NA2 gene ;;

4 - cDNA of the B2M gene after transfection with the genetic construct GDTT1.8NAS3-P4HA2 SEQ ID No: 4, carrying a portion of the P4NA2 gene;

The B2M gene was used as a reference.

It follows from the figure that as a result of transfection with the genetic construct GDTT1.8NAS3-P4HA2 SEQ ID No: 4, the level of specific cDNA of the P4NA2 gene in the primary culture of human endothelial cells from the umbilical vein of HUVEC (ATCC ^® CRL-1730 ™) increased many times.

In FIG. 23

In order to confirm the increase in the amount of prolyl-4-hydroxylase alpha 2 protein in human skin with the introduction of a genetic construct into the skin, an analysis of the change in the quantitative level of protein prolyl 4-hydroxylase alpha 2 in the skin is presented. At the same time, the patient was introduced the PCMV6-XL5 P4NA2 genetic construct SEQ ID No: 5 with the cDNA of the P4NA2 gene (zone B) and a placebo was introduced in parallel, which is a combination of the vector plasmid pCMV6-XL5 without the cDNA of the P4NA2 gene with the transport molecule (zone A) - in skin of the forearm. An increase in the amount of protein in a biopsy sample of the patient’s skin in zone B was shown, into which the genetic construct pCMV6-XL5 P4NA2 SEQ ID No: 5 was introduced, which indicates the effectiveness of this genetic construct.

In FIG. 24

In order to confirm the increase in the amount of prolyl-4-hydroxylase alpha 2 protein in human cartilage with the introduction of the genetic construct VTvaf17-P4HA2 SEQ ID No: 6 with the cDNA of the P4NA2 gene, an analysis of the change in the quantitative level of protein prolyl 4-hydroxylase alpha 2 in cartilage tissue. In this case, the patient was introduced the VTvaf17-P4HA2 genetic construct SEQ ID No: 6 with the cDNA of the P4NA2 gene (zone B) and a placebo, which is a combination of the vector plasmid VTvaf17 without the cDNA of the P4NA2 gene (zone A), was introduced into the cartilage tissue.

An increase in the quantitative level of the protein was shown by shedding of 4-hydroxylase alpha 2 in the lysate of the patient’s cartilaginous tissue biopsy in zone B, into which the genetic construct VTvaf17-P4HA2 SEQ ID No: 6 was introduced with cDNA of the P4NA2 gene, which indicates the effectiveness of this genetic construct.

In FIG. 25

In order to confirm the increase in the amount of prolyl-4-hydroxylase alpha 2 protein in human muscle tissue when the genetic construct GDTT1.8NAS1-P4NA2 of SEQ ID No: 7 with the cDNA of the P4NA2 gene was introduced into this tissue, an analysis of the change in the amount of protein prolyl 4-hydroxylase alpha 2 is presented in muscle tissue.

In this case, the patient was introduced a genetic construct with cDNA of the P4NA2 gene - GDTT1.8NAS1-P4HA2 SEQ ID No: 7 (zone B) and a placebo was introduced in parallel, which is a combination of the vector plasmid GDTT1.8NAS1 not containing the cDNA of the P4NA2 gene with the transport molecule (zone A) - into the muscle tissue of the forearm. An increase in the amount of prolyl-4-hydroxylase alpha 2 protein in a biopsy of the patient’s muscle tissue in zone B was shown, into which the genetic construct GDTT1.8NAS1-P4HA2 SEQ ID No: 7 with cDNA of the P4NA2 gene was introduced, which indicates the effectiveness of this genetic construct.

In FIG. 26

In order to confirm the increase in the amount of prolyl-4-hydroxylase alpha 2 protein to a different individual level when a genetic construct with modified and native P4NA2 gene modified and native cDNA was introduced into the skin of the patients, the quantitative level of prolyl 4-hydroxylase alpha 2 protein in human skin was analyzed depending on the presence and type modifications in the cDNA of the P4NA2 gene.

Each of 32 randomly selected patients was injected into the forearm skin with 7 genetic constructs pCMV6-XL5 P4NA2 SEQ ID No: 1, pCMV6-XL5 P4NA2 SEQ ID No: 2, pCMV6-XL5 P4NA2 SEQ ID No: 3, pCMV6 - XL5 P4NA2 SEQ ID No: 4, pCMV6-XL5 P4NA2 SEQ ID No: 5, pCMV6-XL5 P4NA2 SEQ ID No: 6, pCMV6-XL5 P4NA2 SEQ ID No: 7, and placebo pCMV6-XL5.

Based on the results of the analysis of the amount of protein, shedding of 4-hydroxylase alpha 2 in biopsy samples, we selected indicators for each biopsy sample from each patient, which showed the maximum quantitative levels of protein shed of 4-hydroxylase alpha 2 and combined them into groups based on the following criterion:

In group 1, the maximum amount of protein shedding 4-hydroxylase alpha 2 was observed with the introduction of pCMV6-XL5 P4NA2 SEQ ID No: 1,

in group 2, the maximum amount of protein shedding of 4-hydroxylase alpha 2 was observed with the introduction of pCMV6-XL5 P4NA2 SEQ ID No: 2,

in group 3, the maximum amount of protein shedding 4-hydroxylase alpha 2 was observed with the introduction of pCMV6-XL5 P4NA2 SEQ ID No: 3,

in group 4, the maximum amount of protein shedding of 4-hydroxylase alpha 2 was observed with the introduction of pCMV6-XL5 P4NA2 SEQ ID No: 4,

in group 5, the maximum amount of protein shedding 4-hydroxylase alpha 2 was observed with the introduction of pCMV6-XL5 P4NA2 SEQ ID No: 5,

in group 6, the maximum amount of protein shedding 4-hydroxylase alpha 2 was observed with the introduction of pCMV6-XL5 P4NA2 SEQ ID No: 6,

in group 7, the maximum amount of protein shedding 4-hydroxylase alpha 2 was observed with the introduction of pCMV6-XL5 P4NA2 SEQ ID No: 7.

None of the biopsy specimens showed that the maximum amount of protein shedding 4-hydroxylase alpha 2 was present when a placebo was administered.

Figure 26 shows, for each group of biopsy samples, charts of protein concentration indicators for shedding 4-hydroxylase alpha 2 (averaged within the group if more than one biopsy is included in the group) for all genetic constructs participating in the experiment, after the introduction of these genetic samples into patients constructs containing modified and native cDNA of the P4NA2 gene.

From this example, it follows that the achievement of the maximum amount of protein shed 4-hydroxylase alpha 2 in biopsies of the skin of various patients with the introduction of genetic constructs into the skin, is associated with the individual characteristics of the patients and depends on the presence and type of modifications in the cDNA of the P4NA2 gene included in the genetic construct .

Each genetic construct from the group of genetic constructs is effective in some significant group of patients. Therefore, in order to select the most effective genetic construct from the group of genetic constructs for therapeutic purposes, a preliminary personalized study of the patient is necessary.

Designations:

биоптаты пациентов после введения ГК Р4НА2 SEQ ID No: 1 (A)

biopsy specimens of patients after administration of P4NA2 HA SEQ ID No: 1 (A)

биоптаты пациентов после введения ГК Р4НА2 SEQ ID No: 2 (B)

biopsy specimens of patients after administration of P4NA2 HA SEQ ID No: 2 (B)

биоптаты пациентов после введения ГК Р4НА2 SEQ ID No: 3 (C)

biopsy specimens of patients after administration of P4NA2 HA SEQ ID No: 3 (C)

биоптаты пациентов после введения ГК Р4НА2 SEQ ID No: 4 (D)

biopsy specimens of patients after administration of P4NA2 HA SEQ ID No: 4 (D)

биоптаты пациентов после введения ГК Р4НА2 SEQ ID No: 5 (E)

biopsy specimens of patients after administration of P4NA2 HA SEQ ID No: 5 (E)

биоптаты пациентов после введения ГК Р4НА2 SEQ ID No: 6 (F)

biopsy specimens of patients after administration of P4NA2 HA SEQ ID No: 6 (F)

биоптаты пациентов после введения ГК Р4НА2 SEQ ID No: 7 (G)

biopsy specimens of patients after administration of P4NA2 HA SEQ ID No: 7 (G)

биоптаты пациентов после введения плацебо (Н)

biopsy samples of patients after placebo (N)

In FIG. 27

In order to determine the most effective genetic construct for a particular patient with the P4NA2 cDNA, the quantitative level of the protein shed by 4-hydroxylase alpha 2 in the cell lysates of this patient's fibroblasts transfected with different genetic constructs containing native and modified P4NA2 cDNA was analyzed.

According to the analysis of the amount of protein shedding 4-hydroxylase alpha 2 in the patient’s fibroblast culture, a variant of the genetic construct was isolated, the transfection of which reveals the maximum concentration of the protein shed 4-hydroxylase alpha 2 in the cell culture lysate. In this experiment, the maximum protein concentration shed by 4-hydroxylase alpha 2 in the lysate was observed upon transfection with the pCMV6-XL5 P4NA2 genetic construct SEQ ID No: 4 containing the modified P4NA2 cDNA, as shown in the figure.

Thus, the most effective genetic construct was applied to this patient for subsequent transfection of the patient's cells as part of a therapeutic procedure.

Designations:

1 - cell lysate after transfection with pCMV6-XL5 P4NA2 SEQ ID No: 1 (A)

2 - cell lysate after transfection with pCMV6-XL5 P4NA2 SEQ ID No: 2 (B)

3 - cell lysate after transfection with pCMV6-XL5 P4NA2 SEQ ID No: 3 (C)

4 - cell lysate after transfection with pCMV6-XL5 P4NA2 SEQ ID No: 4 (D)

5 - cell lysate after transfection of PCMV6-XL5 P4NA2 SEQ ID No: 5 (E)

6 - cell lysate after transfection of PCMV6-XL5 P4NA2 SEQ ID No: 6 (F)

7 - cell lysate after transfection of PCMV6-XL5 P4NA2 SEQ ID No: 7 (G)

8 - cell lysate after placebo transfection (H)

In FIG. 28

In order to determine the most effective genetic construct for a particular patient with the P4NA2 cDNA, the amount of prolyl 4-hydroxylase alpha 2 protein was analyzed in the supernatant of this patient’s skin biopsy specimens after the introduction of genetic constructs containing native and modified P4NA2 gene cDNAs.

According to the results of the analysis of the amount of protein shedding 4-hydroxylase alpha 2 in the supernatant of biopsy samples of the patient’s skin, a variant of the genetic construct was isolated, the introduction of which reveals the maximum concentration of the protein prolyl-4-hydroxylase alpha 2 in the biopsy supernatant. In this experiment, the maximum concentration of prolyl-4-hydroxylase alpha 2 protein was noted with the introduction of a genetic construct based on pCMV6-XL5 P4NA2 SEQ ID No: 1 containing native P4NA2 cDNA, as shown in Figure 64.

Thus, the most effective genetic construct for this patient was chosen for its subsequent administration to the patient as part of a therapeutic procedure.

Designations:

1 - supernatant of the biopsy specimen after administration of PCMV6-XL5 P4NA2 SEQ ID No: 1 (A)

2 - supernatant of the biopsy specimen after administration of PCMV6-XL5 P4NA2 SEQ ID No: 2 (B)

3 - supernatant of the biopsy specimen after administration of PCMV6-XL5 P4NA2 SEQ ID No: 3 (C)

4 - supernatant of the biopsy specimen after administration of PCMV6-XL5 P4NA2 SEQ ID No: 4 (D)

5 - supernatant of the biopsy specimen after administration of PCMV6-XL5 P4NA2 SEQ ID No: 5 (E)

6 - supernatant of the biopsy specimen after administration of PCMV6-XL5 P4NA2 SEQ ID No: 6 (F)

7 - supernatant of the biopsy specimen after administration of PCMV6-XL5 P4NA2 SEQ ID No: 7 (G)

8 - supernatant of the biopsy sample after the introduction of placebo (N)

The implementation of the invention.

M, Miinalainen I, Sormunen R, Pakkanen O, Holster T, Soininen R, Prein C, Clausen-Schaumann Н,

A, Tuukkanen J, Kivirikko Kl, Schipani E, Myllyharju J. Severe Extracellular Matrix Abnormalities and Chondrodysplasia in Mice Lacking Collagen Prolyl 4-Hydroxylase Isoenzyme II in Combination with a Reduced Amount of Isoenzyme I. J Biol Chem. 2015.290(27):16964-78.)With a decrease in the expression of genes encoding proteins for the synthesis of prolyl-4-hydroxylase alpha-1 and alpha-2, for example, the P4NA2 gene, a change in the structure of organs and tissues of the body is observed. Despite the fact that mice knocked out with both alleles of the P4NA2 gene (P4HA2 ^{- / -} ) did not show developmental abnormalities, in mice with double knockout for alpha-1 (P4HA1 ^{- / +} ) and alpha-2 (P4HA2 ^{- / -} ) subunits observed growth retardation, moderate chondrodysplasia, kyphosis. At the cellular level, a radical change in the structure of the extracellular matrix was observed (Aro E, Salo AM, Khatri R,

M, Miinalainen I, Sormunen R, Pakkanen O, Holster T, Soininen R, Prein C, Clausen-Schaumann H,

A, Tuukkanen J, Kivirikko Kl, Schipani E, Myllyharju J. Severe Extracellular Matrix Abnormalities and Chondrodysplasia in Mice Lacking Collagen Prolyl 4-Hydroxylase Isoenzyme II in Combination with a Reduced Amount of Isoenzyme I. J Biolm. 2015.290 (27): 16964-78.)

The advantages of using a genetic construct with the P4NA2 gene to correct the expression of the P4NA2 gene and the amount of prolyl-4-hydroxylase alpha 2 protein in the cells of human organs and tissues:

1) it is easier to provide a higher and stable level of the target protein in the cells,

2) transportation of the genetic construct to a wider range of cells of human organs and tissues is provided, as well as more efficient intracellular transportation of the genetic construct.

3) individual characteristics of the patient are taken into account. Thus, to stop the manifestations of human body conditions associated with a decrease in the expression of the P4NA2 gene and / or a decrease in the amount of protein shed by 4-hydroxylase alpha 2, instead of using preparations containing protein shed 4-hydroxylase alpha 2, a genetic construct of the invention was created according to this invention from the group of genetic constructs with one of the modified cDNA of the P4NA2 gene or with a portion of the native unmodified cDNA of the P4NA2 gene encoding the protein shed by 4-hydroxylase alpha 2.

To obtain a genetic construct based on a non-viral vector plasmid, including the cDNA of the P4NA2 gene, to stop the manifestations of human body conditions associated with a decrease in the expression of the P4NA2 gene and / or a decrease in the amount of protein, 4-hydroxylase alpha 2 selected from the group of genetic constructs with cDNA of the P4NA2 gene was shed carry out the following actions:

1. Obtaining a site of unmodified cDNA of the P4NA2 gene containing the protein-coding region of the P4NA2 gene and deletion of 5ʹ untranslated regions and deletion of 3ʹ-untranslated regions, cloning it into an intermediate plasmid, cloning it into expression vector plasmids under the control of eukaryotic regulatory elements for expression of the target gene in this way that the obtained genetic constructs contain a nucleotide sequence encoding the amino acid sequence of a protein prolyl 4-hydroxy lase alpha 2 which is a cDNA R4NA2 unmodified portion of the gene and which is used for further modifications.

2. Introduction of modifications of the P4NA2 gene into the cDNA sequence of the cDNA gene to create a series of cDNA of the P4NA2 gene, providing a sufficient level of protein translation shed by 4-hydroxylase alpha 2 to correct pathological conditions.

3. Cloning of a portion of the unmodified cDNA of the P4NA2 gene and the resulting modified cDNA of the P4NA2 gene into vector plasmids capable of ensuring efficient expression of this cDNA in cells of human organs and tissues.

4. Transformation of each of the obtained genetic constructs of bacterial E. coli cells, analysis of transformed clones for the presence, orientation, and copy number of the cDNA insert and the growth of selected clones to obtain the number of plasmid DNA variants necessary for further work.

5. Isolation of a number of genetic constructs containing modified cDNA of the P4NA2 gene and a portion of the unmodified cDNA of the P4NA2 gene to create a group of highly efficient genetic constructs based on them.

6. Creation of highly efficient genetic constructs, each of which includes a genetic construct with modified cDNA of the P4NA2 gene and a portion of the unmodified cDNA of the P4NA2 gene, or a combination of such a construct with a transport molecule for efficient transfection of various types of cells of organs and tissues and / or introduction into human organs and tissues .

To prove the effectiveness of the created genetic constructs leading to an increase in the expression of the P4NA2 gene, the following studies are carried out:

A) Growing cultures of various types of cells from biopsies of various organs and tissues of a person or using commercial cell lines, for example, skin fibroblasts, or chondroblasts, or keratocytes or epithelial cells of the cornea or endothelial cells from the umbilical vein of a person.

B) Isolation of RNA from a cell culture, for example, skin fibroblasts, or chondroblasts, or keratocytes or epithelial cells of the cornea or endothelial cells from the human umbilical vein, and analysis of the expression of the P4NA2 gene from the genome of these cells.

C) Transfection of a cell culture, for example, skin fibroblasts, or chondroblasts, or keratocytes or epithelial cells of the cornea or keratocytes and epithelial cells of the cornea or endothelial cells from the umbilical vein of a person with genetic constructs containing unmodified and / or modified cDNA of the P4NA2 gene and parallel transfection the same cells with a vector plasmid that does not contain cDNA of the P4NA2 gene in various combinations.

D) Comparative analysis of changes in cDNA levels and / or changes in the amount of protein shed by 4-hydroxylase alpha 2 after transfection in various combinations of cells, for example, skin fibroblasts, or chondroblasts, or keratocytes or epithelial cells of the cornea or endothelial cells from the umbilical vein of a person, genetic constructs containing unmodified and / or modified cDNA of the P4NA2 gene and parallel transfection of the culture of the same cells with a vector plasmid that does not contain cDNA of the P4NA2 gene. This analysis is carried out, namely, before the proposed therapy in order to determine the most effective genetic construct for the patient.

E) Introduction to patients in organs and tissues of a culture of cells with cDNA of the P4NA2 gene, for example, the introduction into the human skin of autologous fibroblasts transfected with a genetic construct with cDNA of the P4NA2 gene and the parallel administration of the cultures of the same cultures to organs and tissues, for example, in the skin cells untransfected and transfected with a vector without inserting the cDNA of the P4NA2 gene and / or introducing patients into organs and tissues of a genetic construct, for example, introducing into the human skin genetic constructs containing unmodified and / or modified bath cDNA of the P4NA2 gene, as well as placebo in various combinations.

F) A comparative analysis of the amount of prolyl-4-hydroxylase alpha 2 protein in human organs and tissues, namely in human skin, after the administration of cells untransfected and transfected with genetic constructs containing the cDNA of the P4NA2 gene and vector plasmids that do not contain the cDNA of the P4NA2 gene and / or after the introduction of genetic constructs containing unmodified or modified cDNA of the P4NA2 gene, as well as a placebo.

G) Introduction to patients in organs and tissues, for example, in the skin, cartilage, or muscle tissue of genetic constructs containing unmodified and / or modified cDNA of the P4NA2 gene, as well as placebo.

H) A comparative analysis of the amount of prolyl-4-hydroxylase alpha 2 protein in human organs and tissues, namely in the skin, cartilage, or human muscle tissue, after the introduction of genetic constructs containing unmodified and / or modified P4NA2 cDNA is also a placebo.

I) Transfection of cell culture, for example, fibroblasts of the skin of patients with genetic constructs containing unmodified and modified cDNA of the P4NA2 gene; introducing patients into organs and tissues, for example, introducing genetic constructs into the patient’s skin containing unmodified and modified cDNA of the P4NA2 gene.

J) A comparative analysis of the amount of prolyl 4-hydroxylase alpha 2 protein in cell lysates, for example skin fibroblast lysates, after transfection of these cells with genetic constructs containing unmodified and modified cDNA of the P4NA2 gene; or in supernatants of biopsy samples of organs and tissues of a patient, for example, in supernatants of skin biopsy samples, after introducing into the skin genetic constructs containing unmodified and modified cDNA of the P4NA2 gene. This analysis is carried out, namely, before the proposed therapeutic procedure in order to select the most effective genetic construct for the patient selected from the group.

Example 1

Obtaining a portion of native (unmodified) cDNA of the P4NA2 gene.

Unmodified cDNA of the P4NA2 gene is a nucleotide sequence homologous to the protein of the coding sequence given in the GenBank database under the number NM_004199; nucleotides 565 through 2172 are translated into the amino acid sequence corresponding to that shown in GenBank under the number NP_004190.1.

Total RNA is obtained from human blood cells using the PAXGeneBlood RNA Kit (Qiagen, Germany) and used to obtain total cDNA by reverse transcription using RevertAid reverse transcriptase (ThermoScientific, USA) and random 9-nucleotide primers according to the method of the manufacturer of reverse transcriptase. Then, using the total cDNA as a template and specific, pre-primed primers complementary to the nucleotides

160-179 5 'GAGCGCTGTGCTGGAAGGGA 3' (P4HA2-F1)

and

2371-2352 5 'GGACAGCAGAGCCAGCACTTGA 3' (P4HA2-R1)

from the P4NA2 mRNA sequence (GenBank NM_004199), a portion of the native (unmodified) P4NA2 cDNA containing the protein-coding region of the P4HA2 gene and partial deletions of 5 'and 3'-untranslated regions is obtained. Polymerase chain reaction (PCR) is carried out using Phusion DNA polymerase (ThermoScientific, USA), giving products with blunt ends. PCR was performed using a Master CyclerGradient amplifier (Eppendorf, USA) in 50 μl. a reaction mixture containing 0.5 μl of total cDNA of the first chain, 0.1 μM of each primer P4NA2 F1 and P4NA2 R1, 250 μM of each deoxynucleotide triphosphate (dATP, dTCP, dTTP and dGTP), 100 mMtris-HCl (pH 8.85 at 20 ° C), 50 mM ammonium sulfate, 250 mM potassium chloride, 0.01% Tween-20 and 5 units. Pfu DNA polymerase (PhusionThermoScientific.USA) under the following conditions: initial denaturation at + 98 ° C for 30 sec., 35 cycles, including denaturation at + 98 ° C for 10 sec., Annealing of primers at + 64 ° C in within 30 sec. and elongation at + 68 ° C for 4 minutes.

The amplification product was isolated from agarose gel using the QIAquickGelExtractionKit kit (Qiagen, Germany)

An amplified 1608 bp cDNA fragment carrying a portion of native (unmodified) P4NA2 cDNA was cloned in pUC19 plasmid vector (NEB, USA, cat. No. N3041S). The DNA of the vector plasmid pUC19 (NEB, USA, cat. No. N3041S) is digested with restriction enzyme restrictase enzyme giving blunt ends, for example, Smal (NEB, USA), and is treated with alkaline phosphatase. The amplified cDNA fragment and the linearized plasmid pUC19 are ligated with 400,000 U / ml phage T4 DNA ligase. (NEB, USA, Cat. No. M0202S) at the rate of 1 μl of the enzyme per 1 μg of DNA. Ligation is carried out in a volume of 20 μl in the presence of 2 mM ATP, 50 mM Tris-HCl, pH 7.6, 10 mM MgCl2, 10 mM DTT for 10 hours at + 160 ° C.

The resulting mixture was transformed with competent E. coliTop10 cells (http://molbiol.ru/protocol/03_04.html), which were plated on Petri dishes with L-agar containing 100 μg / ml ampicillin and 40 μl per cup of solutions of 100 mM IPTG and 2% X-gal. Separate colonies are analyzed for the presence of an insert using PCR with primers P4HA2-F1 and P4HA2-R1 and confirmed by sequencing according to the Sanger method.

Example 2

Obtaining genetic constructs with a site of unmodified cDNA of the P4NA2 gene

To express a portion of the unmodified cDNA of the P4NA2 gene in cells of human organs and tissues, the variant cDNA of the P4NA2 gene according to Example 1 is placed in a vector plasmid, the selection of which uses the following selection criteria:

1) The plasmid must necessarily replicate in E. coli, preferably with a high copy number;

2) The plasmid must contain a bacterial selection factor or another selection factor that does not contain antibiotic resistance genes;

3) The plasmid must contain eukaryotic regulatory elements - promoter and terminator, polyadenylation signal, enhancer and intron (e) element (s);

4) The plasmid must have a convenient polylinker for cloning.

5) The plasmid may contain nucleotide sequences with regulatory elements for replication in mammalian cells, for example, such as SV40 ori from the monkey virus SV40 or ori P / EBNA-1 from the human Epstein-Barr virus.

6) The plasmid may also contain additional regulatory elements that enhance the translation of the protein, for example, binding sites with the transcription factor NFkB, which ensures the active transport of plasmid DNA into the cell nucleus for more efficient transcription of the target gene.

Examples of plasmids that meet these criteria are pCMV6-XL5, pCMV6-Kan / Neo (OriGene, USA), VTvaf17 (Cell and Gene Therapy Ltd, UK), GDTT1.8NAS1, GDTT1.8NAS3, GDTT1.8NAS7 (Genetic Diagnostics and Therapy 21 LTD, UK) or pCDNA 3.1 (+) (ThermoFisher Scientific, USA), or plasmid vectors of viral origin - for example, human adenovirus of the 5th serotype.

When cloning a portion of the unmodified cDNA of the P4NA2 gene in pCDNA 3.1 (+), the coding nucleotide sequence is placed under the control of the human CMV promoter CMV, which is effective in eukaryotic cells, and the bovine growth hormone gene polyadenylation signal. This plasmid also has a bacterial selection factor, the ampicillin resistance gene, and a eukaryotic selection factor, the neomycin resistance gene.

When cloning a portion of the unmodified cDNA of the P4NA2 gene in pCMV6-XL5, pCMV6-Kan / Neo, the coding nucleotide sequence is placed under the control of the human cytomegalovirus CMV promoter, effective in eukaryotic cells, and the polyadenylation signal of the human growth hormone gene. This plasmid also has a bacterial selection factor, the ampicillin resistance gene.

When cloning a portion of the unmodified cDNA of the P4NA2 gene in VTvaf17, the coding nucleotide sequence is placed under the control of the promoter of the human elongation factor gene EF1A with its own enhancer, which is effective in eukaryotic cells and the polyadenylation signal of the human growth factor gene. Also, this plasmid has a selection factor that allows positive selection without the use of antibiotics.

When cloning a portion of the unmodified cDNA to the P4NA2 gene in GDTT1.8NAS1, GDTT1.8NAS3, GDTT1.8NAS7, the coding nucleotide sequence is placed on / to control the promoters of the desmin, endoglin, and nephrin gene promoters, respectively, having their own enhancer and polyadenylation signal from the human growth factor gene. Dunk of the plasmid also has a selection factor that allows positive selection without antibiotics.

To obtain genetic constructs with a plot of unmodified cDNA of the P4NA2 gene, the vector plasmid pUC19-P4HA2 of Example 1 with a plot of unmodified cDNA of P4NA2 containing the protein-coding region of the P4NA2 gene and partial deletions of 5 'and 3'-untranslated regions is used as a template for amplification with the following pairs of primers:

P4NA2 F2 5 'GGAGATCTGCCACCATGAAACTCTGGGTG 3'

P4NA2 R2 5 'CCTCTAGATCAGTCAACTTCTGTTGATCC 3'

Amplification is carried out under the conditions described in Example 1. Next, the amplification products are reprecipitated with ethanol, the precipitates are washed with 70% ethanol and dissolved in 1 × TE buffer, and then subjected to restriction with endonucleases EcoRI and XbaI.

The restriction products are separated by 1% agarose gel electrophoresis, stained with ethidium bromide solution and DNA fragments corresponding to the EcoRI insert gene -P4NA2-XbaI, and the linearized plasmid PCMV6-XL5 and pCMV6-Kan / Neo and pCDNA 3.1 (+), cut out , isolated from the gel using a QIAquickGelExtractionKit gel isolation kit (Qiagen, Germany) and mixed. The resulting mixture of restriction fragments is ligated using T4 phage DNA ligase. Ligation is carried out for 10-15 minutes at room temperature, and then at 12 ° C for 10 hours. Ligation DNA is used to transform E. coli cells according to standard methods using calcium chloride (T. Maniatis et al. Molecular Cloning . M., Mir, 1984). Transformed cells were selected on agar medium LB with ampicillin (100 μg / ml). Plasmid DNA was isolated from ampicillin-resistant colonies using the QiagenSpinMiniprepKit plasmid isolation kit (Qiagen, Germany), analyzed by restriction enzyme digestion with EcoRI and XbaI, and the genetic construct (s) were selected by DNA sequencing of genetic constructs using the Sanger method.

To clone the EcoRI-P4NA2-XbaI insert gene into the vectors VTvaf17 and GDTT1.8NAS1, GDTT1.8NAS3, GDTT1.8NAS7, after extracting it from the gel, 2 additional units are processed. T4 DNA polymerase E.coli and 2 units Act. the Klenov fragment of E. coli polymerase I DNA with the addition of 10 mM dNTP, and then ligated into the VTvaf17 and GDTT1.8NAS1, GDTT1.8NAS3, GDTT1.8NAS7 vectors preliminarily lanered with EcoRM restriction endonuclease. Ligation DNA is used to transform cells of strain E, coli SCS110-AF and E. coli JM-110-NAS by electroporation. To prepare electrocompetent cells of the strain Escherichia coli SCS 110-AF and Escherichia coli JM-110-NAS, a single colony was infected with 10 ml of LB medium and cultured overnight in an orbital shaker at 150 rpm and 37 ° C. The next day, 1/20 is sown in 100 ml of LB medium and cultured in an orbital shaker at 150 rpm and 37 ° C until reaching OD ₆₀₀ = 0.5. Upon reaching the required optical density, the cells are cooled to 0 ° C and centrifuged for 10 min at 4000 g. Next, the medium is removed, the cells are washed from the residues of the medium with ice-cold 100 ml bidistilled water twice, then washed with 20 ml of 10% glycerol. After that, the cells are resuspended in 1 ml of 10% glycerol and used for transformation by electroporation. Electroporation is carried out in 1 mm cuvettes at 2 kV, 200 Ohms, 25 μF on a Gene Pulser Xcell device (Bio-Rad, USA). The pulse time is from 4.9 to 5.1 ms, using 1-10 ng of the vector. Then the cells were cultured for 2.5 hours in SOC medium in a shaker-incubator at 30 ° C. Then, the cells are plated on Petri dishes with agarized selective medium containing yeast extract, peptone, 6% sucrose, and 10 μg / ml chloramphenicol.

Plasmid DNA from the resistant colonies was isolated using the QiagenSpinMiniprepKit plasmid isolation kit (Qiagen, Germany), analyzed by restriction enzyme digestion with BamHI and SaIl, and the genetic construct (s) were selected by DNA sequencing of genetic constructs using the Sanger method.

Bacteria containing the obtained genetic constructs based on the vectors pCMV6-XL5, pCMV6-Kan / Neo and pCDNA 3.1 (+) are grown on LB liquid medium with ampicillin. Bacteria of the Escherichia coli strain SCS110-AF / VTvaf17, Escherichia coli JM-110-NAS / GDTT1.8NAS1, Escherichia coli JM-110-NAS / GDTT1.8NAS3, Escherichia coli JM-110-NAS / GDTT1.8NAS7 containing the obtained genetic constructs based on the vectors VTvaf17, GDTT1.8NAS1, GDTT1.8NAS3, GDTT1.8NAS7, respectively, grown on a selective medium containing yeast extract, peptone, 6% sucrose, as well as 10 μg / ml chloramphenicol, and cultured overnight in an orbital shaker at 150 rpm / min and 37 ° C, lyse and secrete plasmid DNA for further transfection with a special kit for the isolation of plasmids QiagenMaxiKit.

Thus, expression genetic constructs are obtained based on the vectors pCMV6-XL5, pCMV6-Kan / Neo and pCDNA 3.1 (+), VTvaf17, GDTT1.8NAS1, GDTT1.8NAS3, GDTT1.8NAS7 which contain a nucleotide sequence of 1608 bp in length, which includes the protein coding region of the P4HA2 gene and does not contain the untranslated 5 'and 3' regions of the gene with the primary structure shown in FIG. 1 Seq ID No. 1.

Example 3

P4NA2 gene cDNA modification in genetic constructs.

To obtain modified cDNA of the P4NA2 gene in order to increase the efficiency of transcription and translation of the protein, 4-hydroxylase alpha 2 and / or personalized selection of the most effective genetic construct for a particular patient was shed in human tissue and organ cells to the site of the unmodified c4NA sequence of the P4NA2 gene, using codon optimization principle, make modifications by replacing minor codons with synonymous major ones, or carry out amino acid substitutions, or carry out deletions so that modifications do not impair transcription and translation processes.

All modifications are carried out in several stages by PCR mutagenesis using a QuikChP4HA2e II XL kit or QuikChP4HA2e Multi Site-Directed Mutagenesis Kit (AgilentTechnologies, USA,) according to the manufacturer's recommendations. http://www.agilent.com/cs/library/usermanuals/Public/200513.

To the plasmid DNA obtained in Example 2, 50-100 ng of primer, or a double-stranded DNA fragment containing the necessary replacement, insertion or deletion, is added in the amount of 10-50 ng and PCR is carried out in QuikChP4HA2e Multi reaction buffer (Agilent Technologies, USA) with a mixture of QuikChP4HA2e Multi mix enzymes (Agilent Technologies, USA) under the following conditions: 95 ° C, 1 min; further from 30 to 35 cycles: 95 ° С 1 minute, 55 ° С 1 minute, 65 ° С 2 minutes / 1000 bp After PCR, 1-2 μl of restriction enzyme Dpn I was added to the amplification mixture and incubated for 1-2 hours at 37 ° C. The resulting mixture for pCMV6-XL5, pCMV6-Kan / Neo and pCDNA 3.1 (+) transform competent E. coli Tor10 cells (http://molbiol.ru/protocol/03_04.html), which are seeded on plates with L-agar, containing 100 μg / ml ampicillin. Transformed colonies are analyzed for mutation by sequencing plasmid DNA according to the Sanger method. For VTvaf17 and GDTT1.8NAS1, GDTT1.8NAS3, GDTT1.8NAS7, competent cells respectively transform Escherichia coli SCS110-AF (Cell and gene therapy Ltd.) and Escherichia coli JM-110-NAS / GDTT1.8NAS1, Escherichia coli JM- 110-NAS / GDTT1.8NAS3, Escherichia coli JM-110-NAS / GDTT1.8NAS7 (Genetic Diagnostics and Therapy 21 LTD, UK), which are plated on Petri dishes with agarized selective medium containing yeast extract, peptone, 6% sucrose, as well as 10 μg / ml chloramphenicol. Transformed colonies are analyzed for mutation by sequencing plasmid DNA according to the Sanger method.

To obtain the modified cDNA of the P4NA2 gene, SEQ ID No 2, sequential PCR mutagenesis is performed on plasmids VTvaf17-P4HA2 Seq ID No 1, GDTT1.8NAS1-P4NA2 Seq ID No 1, GDTT1.8NAS3-P4HA2 Seq ID No 1, GDTT1.8NAS7 No 1, pCMV6-XL5-P4HA2 Seq ID No 1, pCMV6-Kan / Neo-P4HA2 Seq ID No 1 and pCDNA 3.1 (+) - P4NA2 Seq ID No1 with primers complementary to the native P4NA2 cDNA shown in GenBank under NM_0099 No. :

1. P4NA2 1075-1105 F:

5 'GGGATGGGCCGCTCCGCCTACAATGAAGGGG 3',

Complementary to nucleotides 1075-1105, with substitution G → C at position 1089;

2. P4NA2 1108-1133 F:

5 'TATTATCATACCGTGTTGTGGATGGA 3',

Complementary to nucleotides 1108-1133, with the replacement of G → C at position 1119;

3. P4NA2 1228-1256 F:

5 'GATCTGCACCGGGCCCTGGAGCTCACCC 3',

Complementary to nucleotides 1228-1256, with the replacement of T → G at position 1239;

4. P4NA2 1279-1308 F:

5 'AGCCACGAACGGGCTGGAGGGAATCTGCGG 3',

Complementary to nucleotides 1279-1308, with the replacement of A → G at position 1290;

The nucleotide sequence of the P4NA2 cDNA thus modified SEQ ID No: 2 contains 2 nucleotide substitutions G → C at positions 1089, 1119; 1 nucleotide substitution T → G at position 1239, 1 nucleotide substitution A → G at position 1290; which do not lead to changes in the amino acid sequence of the protein encoded by the sequence of the P4HA2 gene and has a primary structure shown in FIG. 2:

To obtain the modified cDNA of the P4NA2 gene, SEQ ID No 3, sequential PCR mutagenesis is performed on plasmids VTvaf17 Seq ID No 1, GDTT1.8NAS1-P4NA2 Seq ID No 1, GDTT1.8NAS3-P4HA2 Seq ID No 1, GDTT1.8NAS7-P4HA2 No. 1, pCMV6-XL5-P4HA2 Seq ID No. 1, pCMV6-Kan / Neo-P4HA2 Seq ID No. 1 and pCDNA 3.1 (+) - P4NA2 Seq ID No. 1 with primers complementary to the native P4NA2 cDNA shown in GenBank under NM_0099 No. :

1. P4NA2 1075-1105 F:

5 'GGGATGGGCCGCTCCGCCTACAATGAAGGGG 3',

Complementary to nucleotides 1075-1105, with substitution G → C at position 1089;

2. P4NA2 1108-1133 F:

5 'TATTATCATACCGTGTTGTGGATGGA 3',

Complementary to nucleotides 1108-1133, with the replacement of G → C at position 1119;

3. P4NA2 1228-1256 F:

5 'GATCTGCACCGGGCCCTGGAGCTCACCC 3',

Complementary to nucleotides 1228-1256, with the replacement of T → G at position 1239;

4. P4NA2 1279-1308 F:

5 'AGCCACGAACGGGCTGGAGGGAATCTGCGG 3',

Complementary to nucleotides 1279-1308, with the replacement of A → G at position 1290;

5. P4NA2 1336-1365 F:

5 'AGAGAAAAAACCTTAACAAATCAGACAGAA 3',

Complementary to nucleotides 1336-1365, with the replacement of G → C at position 1347;

6. P4NA2 1433-1464 F:

5 'ACGAGAGCCTCTGTCGGGGGGAGAGGGTGTCAAA 3',

Complementary to nucleotides 1433-1464, with the replacement of T → G at position 1449;

7. P4NA2 1463-1489 F:

5 'AACTGACACCCCGGAGACAGAAGAGGC 3',

Complementary to nucleotides 1463-1489, with the replacement of T → G at position 1476;

8. P4NA2 1648-1681 F:

5 'GCACGGGCCACCGTTCGGGATATCACAAGACAGGAG 3',

Complementary to nucleotides 1648-1681, with substitutions A → G at position 1653 and T → G at position 1665;

The nucleotide sequence of the P4NA2 cDNA thus modified SEQ ID No: 3 contains 3 nucleotide substitutions G → C at positions 1089, 1119, 1347; 4 nucleotide substitutions T → G at positions 1239, 1449, 1476, 1665, 2 nucleotide substitutions A → G at positions 1290, 1653; which do not lead to changes in the amino acid sequence of the protein encoded by the sequence of the P4HA2 gene and has a primary structure shown in FIG. 3:

To obtain the modified cDNA of the P4NA2 gene, SEQ ID No. 4, sequential PCR mutagenesis is performed on plasmids VTvaf17-P4NA2 Seq ID No. 1, GDTT1.8NAS1-P4NA2 Seq ID No. 1, GDTT1.8NAS3-P4HA2 Seq ID No. 1, GDTT1.8NAS7 No 1, pCMV6-XL5-P4HA2 Seq ID No 1, pCMV6-Kan / Neo-P4HA2 Seq ID No1 and pCDNA 3.1 (+) - Р4НА2 Seq ID No1 with primers complementary to the native P4NA2 cDNA sections listed in GenBank under NM_004199:

1. P4NA2 1075-1105 F:

5 'GGGATGGGCCGCTCCGCCTACAATGAAGGGG 3',

Complementary to nucleotides 1075-1105, with substitution G → C at position 1089;

2. P4NA2 1108-1133 F:

5 'TATTATCATACCGTGTTGTGGATGGA 3',

Complementary to nucleotides 1108-1133, with the replacement of G → C at position 1119;

3. P4NA2 1228-1256 F:

5 'GATCTGCACCGGGCCCTGGAGCTCACCC 3',

Complementary to nucleotides 1228-1256, with the replacement of T → G at position 1239;

4. P4NA2 1279-1308 F:

5 'AGCCACGAACGGGCTGGAGGGAATCTGCGG 3',

Complementary to nucleotides 1279-1308, with the replacement of A → G at position 1290;

5. P4NA2 1336-1365 F:

5 'AGAGAAAAAACCTTAACAAATCAGACAGAA 3',

Complementary to nucleotides 1336-1365, with the replacement of G → C at position 1347;

6. P4NA2 1433-1464 F:

5 'ACGAGAGCCTCTGTCGGGGGGAGAGGGTGTCAAA 3',

Complementary to nucleotides 1433-1464, with the replacement of T → G at position 1449;

7. P4NA2 1463-1489 F:

5 'AACTGACACCCCGGAGACAGAAGAGGC 3',

Complementary to nucleotides 1463-1489, with the replacement of T → G at position 1476;

8. P4NA2 1648-1681 F:

5 'GCACGGGCCACCGTTCGGGATATCACAAGACAGGAG 3',

Complementary to nucleotides 1648-1681, with substitutions A → G at position 1653 and T → G at position 1665;

9. P4NA2 1750-1777 F:

5 'GCCCGGGTAAATCGGCGGATGCAGCATA 3',

Complementary to nucleotides 1750-1777, with A → G substitutions at position 1755 and T → G at position 1764;

10. P4NA2 1873-1898 F:

5 'GATGAGCGGGATACTTTCAAGCATTT 3',

Complementary to nucleotides 1873-1898, with the replacement of A → G at position 1881;

11. P4NA2 1899-1928 F:

5 'AGGGACCGGGAATCGGGTGGCTACTTTCTT 3',

Complementary to nucleotides 1899-1928, with substitutions G → C at position 1905 and T → G at position 1914

12. P4NA2 2049-2078 F:

5 'TGACTACCGGACAAGACATGCTGCCTGCCC 3',

Complementary to nucleotides 2049-2078, with the replacement of A → G at position 2058;

13. P4NA2 2119-2145 F:

5 'CATGAACGGGGACAGGAGTTCTTGAGA 3',

Complementary to nucleotides 2119-2145, with the replacement of A → G at position 2127;

The nucleotide sequence of the P4NA2 cDNA thus modified SEQ ID No: 4 contains 4 nucleotide substitutions G → C at positions 1089, 1119, 1347, 1905; 6 nucleotide substitutions T → G at positions 1239, 1449, 1476, 1665, 1764, 1914; 6 nucleotide substitutions A → G at positions 1290, 1653, 1755, 1881, 2058, 2127; which do not lead to changes in the amino acid sequence of the protein encoded by the sequence of the P4HA2 gene and has a primary structure shown in FIG. four:

To obtain the modified cDNA of the P4NA2 gene, SEQ ID No. 5, PCR mutagenesis is performed on plasmids VTvaf17-P4HA2 Seq ID No. 1, GDTT1.8NAS1-P4HA2 Seq ID No. 1, GDTT1.8NAS3-P4HA2 Seq ID No. 1, GDTT1.8NAS7-P7 ID No 1, pCMV6-XL5-P4HA2 Seq ID No 1, pCMV6-Kan / Neo-P4HA2 Seq ID No 1 and pCDNA 3.1 (+) - P4NA2 Seq ID No 1 with a synthesized double-stranded DNA fragment of 245 n.p.

The nucleotide sequence of the P4NA2 cDNA thus modified SEQ ID No: 5, length 1602 n.p. has the primary structure shown in FIG. 5.

To obtain the modified cDNA of the P4NA2 gene, SEQ ID No 6, sequential PCR mutagenesis is carried out on plasmids VTvaf17-P4HA2 Seq ID No 1, GDTT1.8NAS1-P4NA2 Seq ID No 1, GDTT1.8NAS3-P4HA2 Seq ID No 1, GDTT1.8NAS7 Seq ID No. 1, pCMV6-XL5-P4HA2 Seq ID No. 1, pCMV6-Kan / Neo-P4HA2 Seq ID No. 1 and pCDNA 3.1 (+) - P4NA2 Seq ID No. 1, first with a 245 bp double-stranded DNA fragment

and then with primers complementary to regions of the native P4NA2 cDNA shown in GenBank under the number NM_004199:

1. P4NA2 1075-1105 F:

5 'GGGATGGGCCGCTCCGCCTACAATGAAGGGG 3',

Complementary to nucleotides 1075-1105, with substitution G → C at position 1089;

2. P4NA2 1108-1133 F:

5 'TATTATCATACCGTGTTGTGGATGGA 3',

Complementary to nucleotides 1108-1133, with the replacement of G → C at position 1119;

3. P4NA2 1228-1256 F:

5 'GATCTGCACCGGGCCCTGGAGCTCACCC 3',

Complementary to nucleotides 1228-1256, with the replacement of T → G at position 1239;

4. P4NA2 1279-1308 F:

5 'AGCCACGAACGGGCTGGAGGGAATCTGCGG 3',

Complementary to nucleotides 1279-1308, with the replacement of A → G at position 1290;

5. P4NA2 1336-1365 F:

5 'AGAGAAAAAACCTTAACAAATCAGACAGAA 3',

Complementary to nucleotides 1336-1365, with the replacement of G → C at position 1347;

6. P4NA2 1433-1464 F:

5 'ACGAGAGCCTCTGTCGGGGGGAGAGGGTGTCAAA 3',

Complementary to nucleotides 1433-1464, with the replacement of T → G at position 1449;

The thus modified nucleotide sequence of the P4NA2 cDNA SEQ ID No: 6, length 1602 n.p. contains 3 nucleotide substitutions G → C at positions 1089, 1119, 1347; 2 nucleotide substitutions T → G at positions 1239, 1449, 1 nucleotide substitution A → G at position 1290; a deletion of 6 nucleotide pairs, as well as a replacement of 8 amino acid residues and has a primary structure shown in FIG. 6:

To obtain the modified cDNA of the P4NA2 gene, SEQ ID No. 7, sequential PCR mutagenesis is performed on plasmids VTvaf17-P4HA2 Seq ID No. 1, GDTT1.8NAS1-P4NA2 Seq ID No. 1, GDTT1.8NAS3-P4HA2 Seq ID No. 1, GDTT1.8NAS7 Seq ID No 1, pCMV6-XL5-P4HA2 Seq ID No 1, pCMV6-Kan / Neo-P4HA2 Seq ID No 1 and pCDNA 3.1 (+) - P4HA2 Seq ID No 1, first with a 245 bp double-stranded DNA fragment

and then with primers complementary to regions of the native P4HA2 cDNA shown in GenBank under the number NM_004199:

1.P4HA2 1075-1105 F:

5 'GGGATGGGCCGCTCCGCCTACAATGAAGGGG 3',

Complementary to nucleotides 1075-1105, with substitution G → C at position 1089;

2.P4HA2 1108-1133 F:

5 'TATTATCATACCGTGTTGTGGATGGA 3',

Complementary to nucleotides 1108-1133, with the replacement of G → C at position 1119;

3.P4HA2 1228-1256 F:

5 'GATCTGCACCGGGCCCTGGAGCTCACCC 3',

Complementary to nucleotides 1228-1256, with the replacement of T → G at position 1239;

4.P4HA2 1279-1308 F:

5 'AGCCACGAACGGGCTGGAGGGAATCTGCGG 3',

Complementary to nucleotides 1279-1308, with the replacement of A → G at position 1290;

5. P4NA2 1336-1365 F:

5 'AGAGAAAAAACCTTAACAAATCAGACAGAA 3',

Complementary to nucleotides 1336-1365, with the replacement of G → C at position 1347;

6. P4NA2 1433-1464 F:

5 'ACGAGAGCCTCTGTCGGGGGGAGAGGGTGTCAAA 3',

Complementary to nucleotides 1433-1464, with the replacement of T → G at position 1449;

7. P4NA2 1463-1489 F:

5 'AACTGACACCCCGGAGACAGAAGAGGC 3',

Complementary to nucleotides 1463-1489, with the replacement of T → G at position 1476;

8. P4NA2 1648-1681 F:

5 'GCACGGGCCACCGTTCGGGATATCACAAGACAGGAG 3',

Complementary to nucleotides 1648-1681, with substitutions A → G at position 1653 and T → G at position 1665;

9. P4NA2 1750-1777 F:

5 'GCCCGGGTAAATCGGCGGATGCAGCATA 3',

Complementary to nucleotides 1750-1777, with A → G substitutions at position 1755 and T → G at position 1764;

10. P4NA2 2049-2078 F:

5 'TGACTACCGGACAAGACATGCTGCCTGCCC 3',

Complementary to nucleotides 2049-2078, with the replacement of A → G at position 2058;

11. P4NA2 2119-2145 F:

5 'CATGAACGGGGACAGGAGTTCTTGAGA 3',

Complementary to nucleotides 2119-2145, with the replacement of A → G at position 2127;

The nucleotide sequence of the P4NA2 cDNA thus modified SEQ ID No: 7, length 1602 n.p. contains 3 nucleotide substitutions G → C at positions 1089, 1119, 1347; 5 nucleotide substitutions T → G at positions 1239, 1449, 1476, 1665, 1764; 5 nucleotide substitutions A → G at positions 1290, 1653, 1755, 2058, 2127; a deletion of 6 nucleotide pairs, as well as a replacement of 8 amino acid residues and has a primary structure shown in FIG. 7:

Thus, genetic constructs (expression vectors) are obtained on the basis of plasmids VTvaf17, GDTT1.8NAS1, GDTT1.8NAS3, GDTT1.8NAS7, pCMV6-XL5, pCMV6-Kan / Neo and pCDNA 3.1 (+) in which a portion of the unmodified cDNA gene was cloned P4NA2 (SEQ ID No: 1) and the modified nucleotide sequences of the cDNA of the P4NA2 gene (SEQ ID No: 2-7) encoding the protein shed by 4-hydroxy lase alpha 2.

Escherichia coli bacteria containing the obtained genetic constructs based on the vectors pCMV6-XL5, pCMV6-Kan / Neo and pCDNA 3.1 (+) are grown on LB liquid medium with ampicillin, lysed, and plasmid DNA is isolated for transfection with a special QiagenMaxiKit plasmid isolation kit (Qiagen , Germany) to obtain the corresponding genetic constructs.

Bacteria of the Escherichia coli strain SCS110-AF / VTvaf17, Escherichia coli JM-110-NAS / GDTT1.8NAS1, Escherichia coli JM-110-NAS / GDTT1.8NAS3, Escherichia coli JM-110-NAS / GDTT1.8NAS7 containing the obtained genetic constructs based on the vectors VTvaf17, GDTT1.8NAS1, GDTT1.8NAS3, GDTT1.8NAS7 grown on a selective medium containing yeast extract, peptone, 6% sucrose, as well as 10 μg / ml chloramphenicol, then lysed and isolated plasmid DNA for transfection with a special kit for transfection isolation of plasmids Endo Free Plasmid Maxi Kit (Qiagen, Germany).

The resulting genetic constructs are used to transfect eukaryotic cells of organs and tissues and / or insertion into human organs and tissues. As control plasmids, the original vector VTvaf17, GDTT1.8NAS1, GDTT1.8NAS3, GDTT1.8NAS7, pCMV6 - XL5, pCMV6-Kan / Neo, or pCDNA 3.1 (+) without the cDNA of the P4NA2 gene is used.

Example 4

Building up the genetic construct in bacterial culture.

To obtain preparative amounts of a vector plasmid containing one of the P4NA2 gene cDNA variants, the E. coli bacterial cells are transformed using the constructs of Examples 2 and 3 — for constructs based on pCMV6 -XL5, pCMV6-Kan / Neo or pCDNA 3.1 (+) (Maniatis T., et al. Molecular Cloning (Moscow, Mir, 1984). For the VTvaf17-based construct, cells of the Escherichia coli SCS110-AF line are transformed. For constructs based on GDTT1.8NAS1, GDTT1.8NAS3, GDTT1.8NAS7, cells of the Escherichia coli JM-110-NAS line are transformed. The resulting bacterial clones containing the construct are grown in the presence of a selection factor necessary for each type of construct - for constructs based on pCMV6 - XL5, pCMV6-Kan / Neo or pCDNA 3.1 (+) - ampicillin, for constructs based on VTvaf17 and GDTT1.8NAS1, GDTT1.8NAS3, GDTT1.8NAS7 - sucrose.

The ligase mixture of examples 2 and 3 is used to transform competent E. coli cells of strain XLblue, SCS110-AF or JM-110-NAS. Preliminary selection of clones containing the P4NA2 gene cDNA insert in the correct orientation and a single copy is carried out by hydrolysis of plasmid DNA with restriction enzymes EcoRI and XbaI or BamHI and SalI and analysis of restriction products by electrophoresis in 1.2% agarose gel. Selected clones are used for preparative growth of plasmid DNA with the aim of further transfection of a fibroblast culture (keratocytes, corneal epithelial cells, umbilical vein endothelial cells, human chondrocytes, etc.).

To obtain preparative amounts of the genetic expression constructs of Examples 2 and 3 in the vectors pCMV6 -XL5, pCMV6-Kan / Neo or pCDNA 3.1 (+), 20 ml of overnight E.coli culture was grown in LB medium with 150 μg / ml ampicillin. 500 ml of LB medium with 100 μg / ml ampicillin were inoculated with this culture; growth was carried out in a shaker-incubator for 16 hours at 37 ° C and 200 rpm.

To obtain preparative quantities of the genetic expression constructs of Examples 2 and 3, the vectors VTvaf17 and GDTT1.8NAS1, GDTT1.8NAS3, GDTT1.8NAS7 were grown on selective medium containing yeast extract, peptone, 6% sucrose, and 10 μg / ml chloramphenicol Plasmid isolation DNA was performed using the EndoFreePlasmidMaxiKit kit (Qiagen, Germany). The yield was 5 mg of DNA.

Example 5

Cultivation of eukaryotic cells, for example, fibroblasts for subsequent transfection with a genetic construct and analysis of P4NA2 gene expression.

For subsequent transfection with a genetic construct containing one of the cDNA variants of the P4NA2 gene according to Example 3, primary cultures of human fibroblasts are grown from biopsy samples of the patient’s skin.

Using a skin biopsy device, Epitheasy 3.5 (MedaxSRL) takes a biopsy sample of the skin from a zone protected from ultraviolet radiation, for example, behind the auricle or from the side inner surface in the area of the elbow joint, with a volume of at least 10 cubic meters. mm, mass - not less than 11 mg. The patient’s skin is pre-washed with sterile saline and anesthetized with lidocaine solution. The primary cell culture was grown on Petri dishes at 37 ° C in an atmosphere containing 5% CO ₂ in DMEM medium with 10% fetal calf serum and ampicillin 100 U / ml. Trypsinization and reseeding is performed every 5 days; for trypsinization, the cells are washed with PBS and incubated for 30 minutes at 37 ° C in a solution containing 0.05% trypsin and 1 mM EDTA. To neutralize trypsin, 5 ml of culture medium was added to the cells and the suspension was centrifuged at 600 rpm for 5 minutes. The growth of fibroblast culture after 4-5 passages is carried out in 75 ml culture bottles in a medium containing 10 g / l DMEM, 3.7 g / l Na ₂ CO ₃ , 2.4 g / l HEPES, 10% fetal serum and 100 U / ml ampicillin . The change of culture medium is carried out every 2 days. The total duration of culture growth did not exceed 25-30 days.

An aliquot containing 10 ⁶ cells was selected from the cell culture.

The part of the fibroblast culture intended for RNA isolation followed by transcription analysis of the P4NA2 gene is centrifuged at 1000 rpm for 20 minutes and washed twice in PBS buffer by centrifugation at 600 rpm for 5 minutes. Then the cells are resuspended in 1.5 ml of the stabilizing reagent RNAIater and stored at 4 ° C for 10 days for subsequent isolation of RNA.

RNA isolation is performed using the RNeasyMiniKit kit. (Qiagen, Germany). The isolated RNA was analyzed spectrophotometrically, measuring the ratio of optical density at 260 and 280 nm, as well as by capillary electrophoresis on a QIAxcel instrument (Qiagen, Germany) using an RNA Qiality Control cartridge. For further work, only those samples are used for which the total amount of isolated RNA was not less than 50 μg RNA, the ratio of D260: D280 was not less than 1.8, and the ratio of the 28S: 18S bands on capillary electrophoresis was not less than 1: 1.

The synthesis of total cDNA of the first chain is carried out using RevertAid reverse transcriptase (Thermo Scientific, USA), according to the manufacturer's recommendations. 1-2 μg of total RNA is used as a template for the synthesis of the first cDNA strand. 100-200 units are added to the reaction mixture for reverse transcription. reverse transcriptase and 10 pM random 9-nucleotide primer.

Example 6

Analysis of the endogenous expression of the P4NA2 gene in a culture of primary fibroblasts

Analysis of the expression of the P4NA2 gene from the fibroblast genome is carried out in order to subsequently correctly determine the gene therapeutic effect after transfection of these cells with genetic constructs from the cDNA of the P4NA2 VTvaf17-P4NA2 gene SEQ ID No: 1.

For analysis, skin fibroblast cell lines, fibroblasts with low expression of the P4HA2 gene, and fibroblasts with normal expression of the P4NA2 gene are used. The cultivation of fibroblasts is carried out according to example 5. For cell transfection, a 6th generation SuperFect Transfection Reagent dendrimer solution (Qiagen, Germany) is used.

Preparation of DNA-dendrimer complexes is carried out according to the manufacturer's method (QIAGEN, SuperFect Transfection Reagent Handbook, 2002) with some changes: 5 μl (with a concentration of 0.5 μg / μl) of solution are added to 15 μl of the dendrimer (with a concentration of 3 μg / μl) each plasmid DNA and mix thoroughly. Then, each DNA mixture with dendrimer is incubated for 5-10 minutes at a temperature of 15-25 ° C.

Next, the medium is carefully selected from the wells of the cell culture plates (without disturbing the cell layer) and the cells are washed with 3 ml of PBS buffer. To the solutions, each of which contained DNA dendrimer complexes, 600 μl of fetal serum DMEM medium was added and these mixtures were transferred to the wells of the cell plates. Cells are incubated with complexes for 2-3 hours at 37 ° C and in an atmosphere containing 5% CO ₂ . Then the medium is carefully removed and the cell layer is washed with 3 ml of PBS buffer. The culture medium is then added and incubated for 24-48 hours at 37 ° C and in an atmosphere containing 5% CO ₂ , after which the cDNA level of the P4NA2 gene and the control cDNA of the B2M gene are estimated using real-time amplification (SYBR GreenRealTimePCR).

Isolation of RNA from fibroblasts and the synthesis of the first cDNA strand are carried out according to example 5.

The isolated RNA for each cell line of fibroblasts transfected with the VTvaf17-P4NA2 genetic construct SEQ ID No: 1 was analyzed by real-time PCR (SYBR GreenRealTimePCR).

For amplification of cDNA (1608 n.p.), specific for the design of VTvaf17-P4NA2 SEQ ID No: 1, use

direct primer P4NA2 -

FA1: TGTGGCCCGAGTAAATCGTC

and

reverse primer

P4NA2 -RA1: AAGTAGCCACACGATTCCCC.

The length of the amplification product is 179 bp As a reference gene, the beta-2-microglobulin B2M gene is used, the expression level of which in fibroblasts is comparable to that normal for the P4NA2 gene.

PCR amplification is performed using the SYBR GreenQuantitect RT-PCR Kit (Qiagen, USA) or another real-time PCR kit in 20 μl amplification mixture containing: 10 μl QuantiTect SYBR Green RT-PCR MasterMix, 2.5 mM magnesium chloride, 0.5 μm of each primer, 5 μl of total RNA. The reaction is carried out on a CFX96 amplifier (Bio-Rad, USA) under the following conditions: 1 reverse transcription cycle at 50 ° C for 30 minutes, denaturation 98 ° C for 15 minutes, then 40 cycles including denaturation 94 ° C for 15 sec. primer annealing at 59 ° C for 30 sec. and elongation of 72 ° C for 30 sec.

As a positive control, the concentration of amplicons obtained by PCR on matrices representing plasmids at known concentrations containing cDNA sequences of the P4NA2 and B2M genes is used. As a negative control, deionized water is used (PCR accumulation curves of the products of negative and positive controls are not shown in FIG. 8 to simplify visualization). The number of PCR products - cDNA of genes P4NA2 and B2M obtained as a result of amplification, is estimated in real time using the software of the Bio-RadCFXManager 2.1 amplifier.

Based on the analysis of the expression of the P4NA2 gene in different fibroblast cultures (with low expression of the P4HA2 gene and with normal expression of the P4NA2 gene), clones were selected from patients of the same age and type of aging, in which the amount of PCR product corresponding to the cDNA of the P4NA2 gene was 10 -20 times lower than that of the B2M gene cDNA. The accumulation curves of specific PCR products are shown in figure 8.

Example 7

Transfection of a fibroblast cell culture with reduced expression of the P4NA2 gene by genetic constructs selected from the group in order to confirm an increase in the expression of the P4NA2 gene in the culture of these cells.

To assess the effectiveness of genetic constructs containing unmodified and modified cDNA of the P4NA2 gene according to examples 2 and 3, each of them was transfected with a primary human fibroblast culture with reduced expression of the P4NA2 gene obtained in example 5.

This fibroblast culture is grown in culture bottles (25 cm ² ) until the cells cover 50-70% of the surface of the bottle. The total number of cells is at least 1 × 10 ⁷ . The grown cells are divided into 8 parts. Each part of the obtained cells is transfected with one genetic construct selected from the group: VTvaf17-P4HA2 SEQ ID No: 1 or VTvaf17-P4HA2 SEQ ID No: 2 or VTvaf17-P4HA2 SEQ ID No: 3 or VTvaf17-P4HA2 SEQ ID No: 4 or VTvaf17 - P4NA2 SEQ ID No: 5 or VTvaf17- P4NA2 SEQ ID No: 6 or VTvaf17-P4NA2 SEQ ID No: 7 in the presence of a transport molecule (e.g., 6th generation SuperFect Transfection Reagent (Qiagen, Germany) and VTvaf17 vector as a control .

A 6-well plate with DMEM medium with 10% fetal calf serum (1.6 ml per well) is seeded with fibroblasts at a rate of 1 × 10 ⁶ cells per well. Cells are incubated for 18 hours at 37 ° C and in an atmosphere containing 5% CO ₂ .

Preparation of DNA-dendrimer complexes and transfection of cell culture is carried out according to example 6.

Isolation of RNA and synthesis of the first cDNA strand is carried out as in Example 5. The cDNA level of the P4NA2 gene and the control cDNA of the B2M gene are estimated using real-time amplification (SYBR GreenRealTimePCR).

For amplification of cDNA (1608 n.p.), specific for the design of VTvaf17-P4NA2 SEQ ID No: 2, use

direct primer

P4NA2 FA1: CTAAACTTGCACGAGCCACC

and reverse primer

P4NA2 -RA1: AGAAGTCGAAGTGCGGTTCAT

The length of the amplification product is 225 bp, the amplification conditions are as for VTvaf17-P4NA2 SEQ ID No: 1.

For amplification of cDNA (1608 n.p.), specific for the design of VTvaf17-P4NA2 SEQ ID No: 3, use

direct primer

P4NA2 FA1: TGTGGCCCGAGTAAATCGTC

and reverse primer

P4NA2 -RA1: AGAAGTCGAAGTGCGGTTCA

The length of the amplification product is 119 bp, the amplification conditions are as for VTvaf17-P4NA2 SEQ ID No: 1.

For amplification of cDNA (1608 n.p.), specific for the design of VTvaf17-P4NA2 SEQ ID No: 4, use

direct primer

P4NA2 FA1: CGGCGGATGCAGCATATCAC

and reverse primer

P4NA2 -RA1: GTCGAAGTGCGGTTCATACTGT

The length of the amplification product is 99 bp, the amplification conditions are as for VTvaf17-P4NA2 SEQ ID No: 1.

For amplification of cDNA (1602 n.p.), specific for the design of VTvaf17-P4NA2 SEQ ID No: 5, use

direct primer

P4NA2 FA1: CCTGTTGTGGCCCGAGTAAAT

and reverse primer

P4NA2 -RA1: GCTGTCAAAAGGTCGCCTAGA

The length of the amplification product is 144 nucleotides, the amplification conditions, as for VTvaf17-P4NA2 SEQ ID No: 1.

For amplification of cDNA (1602 n.p.), specific for the design of VTvaf17-P4NA2 SEQ ID No: 6, use

direct primer

P4NA2 FA1 GCCCGAGTAAATCGTCGGA

and reverse primer

P4NA2 -RA1 GCTGTCAAAAGGTCGCCTAGA

The length of the amplification product is 135 bp, the amplification conditions are as for VTvaf17-P4NA2 SEQ ID No: 1.

For amplification of cDNA (1602 n.p.), specific for the design of VTvaf17-P4NA2 SEQ ID No: 7, use

direct primer

P4NA2 FA1 ACTTCGACTTCTCTAGGCGAC

and reverse primer

P4NA2 -RA1 TTCTTAGGCCAAATTGCAGCC

The length of the amplification product is 145 nucleotides, the amplification conditions, as for VTvaf17-P4NA2 SEQ ID No: 1.

As a reference gene, the beta-2-microglobulin B2M gene is used, the expression level of which in fibroblasts is comparable to that normal for the P4NA2 gene. As a positive control, the concentration of amplicons obtained by PCR on matrices representing plasmids at known concentrations containing cDNA sequences of the P4NA2 and B2M genes is used. As a negative control, deionized water is used (PCR accumulation curves of the products of negative and positive controls are not shown in FIG. 8 to simplify visualization). The number of PCR products - cDNA of genes P4NA2 and B2M obtained as a result of amplification, is estimated in real time using the software of the Bio-RadCFXManager 2.1 amplifier. The results of the analysis are shown in figures 9-15.

From the graphs it follows that in the case of transfection with a vector without inserting a cDNA of the P4NA2 gene, the level of cDNA of the P4NA2 gene in fibroblasts did not change, and in the case of transfection with genetic constructs selected from the group, the cDNA level of fibroblasts with reduced expression of the P4NA2 gene significantly increased.

It was shown that in each case in cells transfected with genetic constructs VTvaf17-P4NA2 SEQ ID No: 1 or VTvaf17-P4HA2 SEQ ID No: 2 or VTvaf17-P4HA2 SEQ ID No: 3 or VTvaf17-P4HA2 SEQ ID No: 4 or VTvaf17-P4HA2 SEQ ID No: 5 or VTvaf17-P4HA2 SEQ ID No: 6 or VTvaf17-P4HA2 SEQ ID No: 7 there is an increase in the expression of the target gene P4NA2.

Example 8

Transfection of a fibroblast cell culture with normal expression of the P4NA2 gene by a genetic construct with cDNA of the P4NA2 gene in order to confirm the increase in the amount of protein shed by 4-hydroxylase alpha 2 in the culture of these cells.

To assess the change in the amount of 4-hydroxylase alpha 2 shed, we used a human skin fibroblast culture according to Example 5, the 6th generation SuperFect Transfection Reagent dendrimers (Qiagen, Germany) as a transport molecule, and an aqueous solution of dendrimers without plasmid DNA as a control ( A), the vector plasmid pCDNA 3.1 (+) that does not contain the cDNA of the P4NA2 gene (B), as transfecting agents - a genetic construct based on the vector plasmid pCDNA 3.1 (+) containing the cDNA of the P4NA2 gene - pCDNA 3.1 P4NA2 SEQ ID No: 2 (C) Preparation of a DNA Dendrimer and Transf Complex The fibroblast projection was performed according to Example 7.

After transfection, 0.1 ml of 1N HCl was poured into 0.5 ml of culture liquid, mixed thoroughly and incubated for 10 minutes at room temperature. The mixture was then neutralized by adding 0.1 ml of 1.2N NaOH / 0.5M HEPES (pH 7-7.6) and mixed thoroughly.

The supernatant was taken and used to quantify the target protein. Quantitative determination of the P4NA2 gene cDNA product was carried out by enzyme-linked immunosorbent assay (ELISA) using the Human P4NA2 ELISA Kit Prolyl 4-hydroxylase subunit alpha-2, ELISA Kit (# MBS2885550) (MyBioSource, USA), according to the manufacturer's method https: // www .mybiosource.com / prods / ELISA-Kit / Human / Prolyl-4-hydroxylase-subunit-alpha-2 / P4HA2 / datasheet.php? products id = 2885550. The optical density was measured at a wavelength of 450 nm using a ChemWell automatic biochemical and enzyme immunoassay analyzer (Awareness TechNology Inc., USA) and is proportional to the concentration of protein in the sample. The numerical value of the concentration is determined using a calibration curve constructed from calibrators with a known protein concentration of the P4NA2 gene, which is part of the kit. The difference between the optical density (OD) values of the calibrators supplied in triplicate did not exceed 10%.

Statistical processing of the results was carried out using software for statistical processing and visualization of data R, version 3.0.2 (https://www.r-project.org/). Thus, it was shown that transfection of fibroblasts with the obtained genetic construct pCDNA 3.1 P4NA2 SEQ ID No: 2, carrying the cDNA of the P4NA2 gene, leads to an increase in the amount of protein shed by 4-hydroxylase alpha 2 in fibroblast cells. The results are shown in figure 16.

Example 9

The introduction of a fibroblast cell culture transfected with a genetic construct with cDNA of the P4NA2 gene into human skin in order to confirm an increase in the amount of protein shed 4-hydroxylase alpha 2 in a biopsy of human skin.

In order to analyze the change in the amount of protein, 4-hydroxylase alpha 2 was shed in human tissues, three variants of autologous fibroblast culture were introduced into the skin of the forearm - non-transfected (zone A), transfected with a vector without an insert, for example, VTvaf17 (zone B) and transfected with the genetic construct VTvaf17 -P4NA2 SEQ ID No: 7 (zone C) in combination with transport molecules - TRANSFAST TM Transfection Reagent liposomes (PROMEGA, USA).

The suspension of liposomes was prepared according to the manufacturer's method (Promega, TransFast Transfection Reagent, Instruction for Use of Product, E2431) with some changes: 0.4 ml of Nuclease-Free sterile water was added to 0.4 mg of liposome powder. The suspension was mixed well by shaking. The resulting intermediate product of the suspension of liposomes was kept for 18 hours at a temperature of -20 ° C, then thawed at room temperature, resuspended by shaking or on a table mixer type "VORTEX".

To prepare the DNA liposome complex, 400 μl of plasmid DNA solution (with a concentration of 150 μg / ml) was added to 400 μl of liposome solution (with a concentration of 1 mg / ml) and mixed thoroughly. Then the mixture of DNA with liposomes was incubated for 5-10 minutes at a temperature of 15-25 ° C and was used further as a genetic construct.

For subsequent transfection of cells with a genetic construct, primary cultures of human fibroblasts were grown from biopsy samples of the skin of patients according to Example 5.

For transfection, the medium was carefully selected from the wells of a cell culture plate (without disturbing the cell layer) and the cells were washed with 3 ml of PBS buffer. To a solution containing DNA liposome complexes, 600 μl of fetal serum DMEM medium was added and the mixture was transferred to the wells of a cell plate. Cells were incubated with complexes for 2-3 hours at 37 ° C and in an atmosphere containing 5% CO ₂ . Then the medium was carefully removed and the cell layer was washed with 3 ml of PBS buffer. Then, the culture medium was added and incubated for 24-48 hours at 37 ° C in an atmosphere containing 5% CO ₂ .

Part of the cell culture was centrifuged at 700 rpm, the cells were washed twice with physiological saline, resuspended in physiological saline at the rate of 1 million cells in 200 μl and used for administration to the patient. The introduction was carried out by the tunnel method with a 30G needle to a depth of 3 mm. Foci of introducing fibroblast cultures were located at a distance of 3-5 cm from each other.

Determination of the amount of protein shed by 4-hydroxylase alpha 2 was carried out in lysates of biopsy samples of the patient’s skin according to the method described in Example 8.

Biopsy samples were taken 3 days after the introduction of a suspension of fibroblasts. A biopsy was performed from the injection sites of the fibroblast suspension, as well as from the intact skin, using an Epitheasy 3.5 skin biopsy device (MedaxSRL). The patient’s skin was pre-washed with sterile saline and anesthetized with lidocaine solution. The volume of the biopsy sample is not less than 10 cubic meters. mm, mass - not less than 11 mg. The sample was placed in a buffer solution containing 50 mM Tris-HCl pH 7.6, 100 mM NaCl, 1 mM EDTA and 1 mM phenylmethylsulfonyl fluoride, and homogenized to obtain a uniform suspension. The resulting suspension was centrifuged for 10 minutes at 14,000 rpm. The supernatant was taken and used to quantify the target protein.

As can be seen from figure 17, in the skin of the patient in the area of the introduction of fibroblasts transfected with the genetic construct VTvaf17-P4NA2 SEQ ID No: 7, there was an increase in the amount of protein shed by 4-hydroxylase alpha 2, which may indicate increased expression of the P4NA2 gene. Whereas with the introduction of control cultures of fibroblasts (untransfected and transfected with a vector without inserting the cDNA of the P4NA2 VTvaf17 gene), the amount of 4-hydroxylase alpha 2 shed did not change in the skin.

Thus, the effectiveness of the genetic construct when introducing a cell culture of fibroblasts transfected with a genetic construct carrying a modified cDNA of the P4NA2 gene was shown to be increased, which leads to an increase in the expression level of the P4NA2 gene, namely, an increase in the amount of protein shed by 4-hydroxylase alpha 2 in the skin in the injection zone.

Example 10

Transfection of fibroblast cultures from biopsies of a group of different patients with genetic constructs containing modified cDNA of the P4NA2 gene and a portion of the native unmodified cDNA of the P4NA2 gene.

In order to confirm the individual character of increasing the expression of the P4NA2 gene to a different level during transfection of cell cultures of fibroblasts of patients with genetic constructs with modified cDNA of the P4NA2 gene and a portion of the native cDNA of the P4NA2 gene, the quantitative level of protein in cell lysates of fibroblasts obtained from biopsy samples of a group of patients transfected with different genetic constructs was analyzed examples 2 and 3 containing modified cDNA of the P4NA2 gene and a portion of native unmodified cDNA ene R4NA2 depending on the presence and type of modification in the cDNA R4NA2 gene.

The sequence of the site of the native unmodified cDNA of the P4NA2 gene is shown in Figure 1, SEQ ID No: 1., the sequences of the modified cDNA of the P4NA2 gene are shown in Figures 2-7 (SEQ ID No: 2, SEQ ID No: 3, SEQ ID No: 4, SEQ ID No: 5, SEQ ID No: 6, SEQ ID No: 7).

Fibroblast cultures were grown from the same skin biopsy samples taken from the zone of the inner lateral surface in the zone of the elbow joint or from the biopsy sample obtained during cosmetic surgeries 50 patients randomly selected in Example 5, aliquots were selected and the level of protein shed by 4-hydroxylase alpha 2 was evaluated in cell lysates. A cell pellet corresponding to 1 × 10 ⁷ cells was washed with phosphate buffer and resuspended in ice in a lysis buffer containing 25 mM HEPES pH 7.9, 100 MMNaCl, 1 mM EDTA, 1% Triton X-100, 10% glycerol and 1 mM phenylmethylsulfonyl fluoride. The lysate was centrifuged for 5 minutes at 14,000 rpm, the protein concentration in the supernatant was determined by the Bradford method ([Bradford M.M. (1976) Anal. Biochem. 72: 248-254.]).

Then, each of the 50 fibroblast cultures was divided into 27 parts.

One part labeled (A) was transfected according to Example 7 with the pCMV6-XL5 genetic construct SEQ ID No: 1 containing unmodified cDNA of the P4HA2 gene (SEQ ID No: 1.) according to Example 2 in combination with the dendrimer transport molecule of Example 7.

The second part, designated (B), was transfected according to Example 7 with the pCMV6-XL5 genetic construct SEQ ID No: 2 containing a variant of the modified cDNA of the P4NA2 gene (SEQ ID No: 2.) according to Example 3 in combination with the transport molecule dendrimer according to Example 7 .

The 3rd part, designated (C), was transfected according to Example 7 with the pCMV6-XL5 genetic construct SEQ ID No: 3 containing a variant of the modified cDNA of the P4NA2 gene (SEQ ID No: 3.) according to Example 3 in combination with a transport molecule dendrimer according to example 7.

The 4th part, labeled (D), was transfected according to Example 7 with the genetic construction of the pCMV6-XL5 vector SEQ ID No: 4 containing a variant of the modified cDNA of the P4NA2 gene (SEQ ID No: 4.) according to Example 3 in combination with a transport molecule - dendrimer as in example 7.

The 5th part, designated (E), was transfected according to Example 7 with the pCMV6-XL5 genetic construct SEQ ID No: 5 containing a variant of the modified cDNA of the P4NA2 gene (SEQ ID No: 5.) according to Example 3 in combination with a transport molecule dendrimer according to example 7.

The 6th part, labeled (F), was transfected according to Example 7 with the pCMV6-XL5 genetic construct SEQ ID No: 6 containing a variant of the modified P4NA2 gene cDNA (SEQ ID No: 6) according to Example 3 in combination with the transport molecule dendrimer according to Example 7.

The 7th part, designated (G), was transfected according to Example 7 with the pCMV6-XL5 genetic construct SEQ ID No: 7 containing a variant of the modified P4NA2 gene cDNA (SEQ ID No: 7) according to Example 3 in combination with the transport molecule dendrimer according to Example 7.

The 8th part, designated (H), was transfected according to Example 7 with the vector plasmid pCMV6-XL5 that did not contain the cDNA of the P4NA2 gene in combination with the transport molecule dendrimer.

The quantitative determination of the cDNA product of the P4NA2 gene was carried out according to the method described in Example 8.

Based on the results of determining the quantitative level of the protein, 4-hydroxylase alpha 2 was shed, selected indicators for each part of the cell culture from each patient, which showed the maximum quantitative levels of protein and combined them into groups based on the following criterion:

In group 1, the maximum amount of protein was shed by 4-hydroxylase alpha 2, was observed during transfection of unmodified cDNA of the P4NA2 gene SEQ ID No: 1. This group included 11 out of 50 cultures.

In group 2, the maximum amount of protein was shed by 4-hydroxylase alpha 2, was observed during transfection of the modified cDNA of the P4NA2 gene SEQ ID No: 2. This group included 5 out of 50 cultures.

In group 3, the maximum amount of protein was shed by 4-hydroxylase alpha 2; it was observed upon transfection of the modified cDNA of the P4NA2 gene SEQ ID No: 3. This group included 8 out of 50 cultures.

In group 4, the maximum amount of protein was shed by 4-hydroxylase alpha 2, which was observed upon transfection of the modified cDNA of the P4NA2 gene SEQ ID No: 4. This group included 5 out of 50 cultures.

In group 5, the maximum amount of protein was shed by 4-hydroxylase alpha 2, which was observed during transfection of the modified cDNA of the P4NA2 gene SEQ ID No: 5. This group included 6 out of 50 cultures.

In group 6, the maximum amount of protein was shed by 4-hydroxylase alpha 2, which was observed during transfection of the modified cDNA of the P4NA2 gene SEQ ID No: 6. This group included 8 out of 50 cultures.

In group 7, the maximum amount of protein was shed by 4-hydroxylase alpha 2; it was observed upon transfection of the modified cDNA of the P4NA2 gene SEQ ID No: 7. This group included 7 out of 50 cultures.

None of the cell cultures transfected with the genetic construct with a vector plasmid that does not contain the cDNA of the P4NA2 gene showed a maximum quantitative level of the protein shed by 4-hydroxylase alpha 2, in contrast to cell cultures transfected with the genetic constructs with cDNA of the P4NA2 gene.

Figure 18 shows a table of indicators of the amount of protein shed by 4-hydroxylase alpha 2, (averaged over the group if more than one cell culture is included in the group), for all genetic constructs participating in the experiment and for each group of cell cultures after transfection of these cell cultures with genetic constructs containing modified cDNA of the P4NA2 gene and a portion of the native cDNA of the P4NA2 gene.

From this example, it follows that the achievement of the maximum amount of protein was shed by 4-hydroxylase alpha 2 in the skin fibroblast cultures of various patients during their transfection with genetic constructs, is associated with individual characteristics of the patients and depends on the presence and type of modifications in the P4NA2 gene cDNA.

Each genetic construct from the group is effective in some significant group of patients. Therefore, in order to select the most effective genetic construct from a group for therapeutic purposes, a preliminary personalized study of the patient’s biopsy samples, or cells grown from these biopsy samples for maximum therapeutic effect, created by the genetic structures within the group, is necessary.

Example 11

Transfection of the cell culture of keratocytes and epithelial cells of the eye with a genetic construct without a transport molecule in order to confirm the increase in the expression of the P4NA2 gene in these cell cultures.

In order to determine the effectiveness of the genetic construct containing P4NA2 cDNA in various cell types, keratocytes and epithelial cells of the human cornea were transfected in combination with TRANSFAST TM Transfection Reagent transport molecules (PROMEGA, USA) according to Example 8.

Biopsy material from the limb region in the form of a circle with a diameter of 1-1.5 mm was placed in a Petri dish in a balanced Hanks saline solution, dispase (20 μl 25 u / ml) and gentamicin were added and incubated for 24 hours at +4. Then the epithelial layer was separated from the stroma and both tissues were cut into pieces of 1-2 mm3.

A suspension of epithelial cells was obtained by incubating pieces of tissue in a solution of Trypsin-EDTA for 15 minutes at + 37 ° C. Trypsinization was stopped by the addition of a soybean trypsin inhibitor in PBS. Cell suspensions were washed by centrifugation at 700 rpm and resuspended in serum-free DMEM with gentamicin. Cells were grown in 25 cm2 vials with 5 ml of medium at 37 ° C and in an atmosphere containing 5% CO2. After 24 hours, non-adherent cells were removed and 5 ml of fresh medium was added. Reseeding was performed every 7 days in a ratio of 1: 2-1: 3.

A keratocyte suspension was obtained by incubation in RPMI-1640 medium with collagenase 300 u / ml for 2 hours. Then the cells were washed and resuspended in DMEM medium with 20% calf serum and antibiotic-antimycotic. Cells were grown in 25 cm2 vials with 1 ml of medium at + 37 ° C and in an atmosphere containing 5% CO2. Reseeding was performed every 7 days in a ratio of 1: 4.

Since the efficiency of keratocyte transfection is low, we used a genetic construct based on the plasmid pCMV6-Kan / Neo P4NA2 SEQ ID No: 2 containing an additional selection factor, the neor gene, which confers antibiotic resistance to geneticin.

The genetic construct was added to the cells and the cells were incubated for 1 hour at 37 ° C. After incubation, serum-free DMEM medium was added to the epithelial cells and DMEM medium containing 10% fetal calf serum to keratocytes was continued and incubated for 24 hours. After a 24-hour incubation, the cells were seeded in a new DMEM medium containing 400 μg / mL of the selection factor antibiotic G418 (Geneticin). Surviving cells were cultured for 2 weeks in 25 cm3 vials with 5 ml of G418 medium. A total of 24 samples of cultures of epithelial cells and 24 samples of cultures of keratocytes were grown.

An aliquot of 500 μl of suspension was taken from each culture every 24 hours to isolate RNA and analyze the level of specific cDNA. RNA isolation was performed using the RNeasyMiniKit kit (Qiagen, Germany). RNA samples were grouped into 3 groups of 8 samples with a similar concentration and combined. The analysis of the level of specific cDNA of the P4NA1 gene was carried out using real-time amplification according to the procedure and with the primers of Example 6 using the iTaqUniversalSYBRGreenSupermix reagent kit (Bio-Rad, USA), CFX96 amplifier (Bio-RadUSA) and Bio-RadCFXManager 2.1 software. The maximum increase in the expression (transcription) of the P4NA2 gene was observed on day 3 for epithelial cells and on day 5 for keratocytes.

Figure 19 shows the accumulation curves of a specific amplification product corresponding to the cDNA of the P4NA2 gene in keratocytes and epithelial cells of the cornea.

It was shown that in keratocytes and epithelial cells of the human cornea transfected with the pCMV6-Kan / Neo P4NA2 SEQ ID No: 2 genetic construct, in combination with the transport molecules TRANSFAST TM Transfection Reagent liposomes, the expression of the target P4NA2 gene is enhanced.

Example 12

Transfection of the primary culture of smooth muscle cells of the human bladder with a genetic construct with a transport system in the form of Lipofectamine 3000 liposomes (ThermoFisher Scientific, USA) in order to confirm the increase in the expression of the P4NA2 gene in these cell cultures.

In order to determine the effectiveness of the genetic construct containing P4NA2 cDNA in various cell types, the HBdSMC (ATCC PCS-420-012) human primary bladder smooth muscle cell primary culture cells were transfected with the genetic construct using lipofectamine 3000 liposome transport molecules (ThermoFisher Scientific, USA ) The experiment used a genetic construct based on the plasmid GDTT1.8NAS1 P4NA2 SEQ ID No: 3. Cells of the primary culture of smooth muscle cells of the human bladder HBdSMC (ATCC PCS-420-012) were grown in the medium included in the Vascular Smooth Muscle Cell Growth Kit (ATCC ^® PCS-100-042 ™) in an atmosphere of 5% CO ₂ at 37 ° C. To obtain 90% confluency, 24 hours before transfection, cells are seeded in a 24-well plate at the rate of 5 * 10 ⁴ cells / well.

Transfection with the genetic construct GDTT1.8NAS1-P4NA2 SEQ ID No: 3 is carried out as follows. In test tube No. 1, 1 μl of the genetic construct solution (concentration 500 ng / μl) and 1 μl of P3000 reagent were added to 25 μl of Opti-MEM medium (Gibco). Gently mix with gentle shaking. In tube No. 2, 1 μl of Lipofectamine 3000 solution is added to 25 μl of Opti-MEM medium (Gibco). Gently mix with gentle shaking. The contents of tube No. 1 are added to the contents of tube No. 2, incubated for 5 min at room temperature. The resulting solution was added dropwise to the cells in a volume of 40 μl.

As a control, human bladder cells HBdSMC are used, transfected with the GDTT1.8NAS1 DNA vector that does not contain the insert of the target P4NA2 gene. The preparation of the control vector GDTT1.8NAS1 for transfection is carried out as described above.

Isolation of total RNA from transfected cells and PCR amplification is carried out as in Example 6. The beta-2-microglobulin B2M gene is used as the reference gene. As a negative control, deionized water is used. The number of PCR products - cDNA of genes P4NA2 and B2M obtained as a result of amplification, is estimated in real time using the software of the Bio-RadCFXManager 2.1 amplifier.

In order to confirm the increase in the expression of the P4HA2 gene in human HBdSMC cells of the human bladder after transfection of these cells with the genetic construct GDTT1.8NAS1-P4HA2 P4HA2 SEQ ID No: 3 in FIG. 20 shows the graphs of the accumulation of PCR products corresponding to:

1 - cDNA of the P4NA2 gene after transfection with the vector GDTT1.8NAS1;

2 - cDNA of the P4NA2 gene after transfection with the genetic construct GDTT1.8NAS1 P4NA2 SEQ ID No: 3, carrying the portion of the P4NA2 gene;

3 - cDNA of the B2M gene after transfection with the vector GDTT1.8NAS1

4 - cDNA of the B2M gene after transfection with the genetic construct GDTT1.8NAS1 P4NA2 SEQ ID No: 3 carrying the region of the P4NA2 gene;

From the figure it follows that as a result of transfection of human bladder cells with HBdSMC genetic construct GDTT1.8NAS1 P4NA2 SEQ ID No: 3, the level of specific mRNA of the human P4NA2 gene increased many times.

Example 13

Transfection of the cell culture of chondroblasts with a genetic construct with cDNA of the P4NA2 gene in order to confirm the increase in the expression of the P4NA2 gene in these cell cultures.

In order to determine the effectiveness of the genetic construct containing the cDNA of the P4NA2 gene in various cell types, as well as to assess the effect of the transport molecule on the success of transfection with this genetic construct, human chondroblasts were transfected using amphiphilic block copolymers as the transport molecule.

Biopsies from the spine of human embryos of 12 weeks of age weighing 50-150 mg were crushed into fragments up to 10 mm3 and incubated in a buffer solution with collagenase, hyaluronidase and trypsin for 48 hours at + 37 ° C. Cells were washed with serum-free DMEM medium, resuspended in the same medium with gentamicin and grown at + 37 ° C and in an atmosphere containing 5% CO2 in 75 cm2 vials with 15 ml of medium. Growth was carried out in a serum-free medium, since in a monolayer culture, chondrocytes change their shape and biochemical properties. Reseeding was performed every 4 days in a ratio of 1: 3, the cells were removed from the surface of the vial with trypsin-EDTA solution with 0.02% Versen solution.

Amphiphilic block copolymers were used to transfect cells with the genetic construct of the P4NA2 cDNA gene. The methodology was applied according to (IntJPharm, 2012, 427, 80-87) or in (Macromol. Biosci.2011, 11, 652-661), with some changes. The block copolymer was synthesized from a mixture of linear polyethyleneimine (PEI) (Polyscience Inc., USA) and bifunctional polyethylene glycol (PEG) N-hydroxysuccinimidyl-75-N- (3-maleimidopropionyl) -amido-4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73 - tetracosoxapenta-heptacontanoate (MAL-dPEG ™ -NHS ester, Quanta BioDesign, Ltd., USA) in borate buffer. A polyplex was prepared 1 hour before the introduction into the cells by mixing the block copolymer solution with DNA of the pCMV6-Kan / Neo P4NA2 genetic construct SEQ ID No: 3 with modified P4NA2 cDNA SEQ ID No: 3. The transfection mixture was added to the cell suspension in 1 ml DMEM culture medium with 10% fetal serum and ampicillin.

Since a construct containing a selection factor (resistance to geneticin) was used for transfection, the cells after transfection were grown in 25 cm ³ bottles with 5 ml of medium with geneticin for 12 days, changing the medium every 4 days. An aliquot of 500 μl of suspension was taken from each culture every 24 hours to isolate RNA and analyze the level of specific cDNA. RNA isolation was performed using the RNeasyMiniKit kit (Qiagen, Germany). RNA samples were grouped into 3 groups of 8 samples with a similar concentration and combined. The analysis of the level of specific P4NA2 cDNA was performed using real-time amplification according to the procedure and with the primers of Example 6 using the iTaqUniversalSYBRGreenSupermix reagent kit (Bio-Rad, USA), CFX96 amplifier (Bio-Rad, USA) and Bio-RadCFXManager 2.1 software . The maximum increase in P4NA2 transcription was observed on day 5. The figure 21 shows the accumulation curves of a specific amplification product corresponding to the cDNA of the P4NA2 gene.

It was shown that in chondroblasts transfected with the pCMV6-Kan / Neo P4NA2 genetic construct SEQ ID No: 3, using amphiphilic block copolymers as the transport molecule, the expression of the target P4NA2 gene is enhanced.

Example 14

Transfection of the endothelial cell culture from the human umbilical vein HUVEC (ATCC ^® CRL-1730 ™) with a genetic construct with the Lipofectamine 3000 transport system (ThermoFisher Scientific, USA) in order to confirm the increase in P4NA2 gene expression in these cell cultures.

In order to determine the effectiveness of the genetic construct containing P4NA2 cDNA in various cell types, the primary construct of human endothelial cells from human umbilical vein HUVEC (ATCC ^® CRL-1730 ™) was transfected with the genetic construct using the Lipofectamine 3000 transport molecules (ThermoFisher Scientific, USA) with the genetic construct. . The experiment used a genetic construct based on the plasmid GDTT1.8NAS3-P4HA2 P4NA2 SEQ ID No: 4.

The HUVEC primary umbilical vein endothelial cell culture line (ATCC ^® CRL-1730 ™) is grown in F-12K Medium (ATCC) supplemented with 0.05 mg / ml ascorbic acid, 0.01 mg / ml insulin, 0.01 mg / ml transferrin, 10 ng / ml sodium selenite, 0.03 mg / ml Endothelial Cell Growth Supplement (ECGS), 10% serum of cow embryos in an atmosphere of 5% CO ₂ at 37 ° C. To obtain 90% confluency, 24 hours before transfection, cells are seeded in a 24-well plate at the rate of 5 * 10 ⁴ cells / well.

Transfection with the genetic construct GDTT1.8NAS3-P4NA2 P4NA2 SEQ ID No: 4 expressing the human P4NA2 gene is carried out as follows. In test tube No. 1, 1 μl of the genetic construct solution (concentration 500 ng / μl) and 1 μl of P3000 reagent were added to 25 μl of Opti-MEM medium (Gibco). Gently mix with gentle shaking. In tube No. 2, 1 μl of Lipofectamine 3000 solution is added to 25 μl of Opti-MEM medium (Gibco). Gently mix with gentle shaking. The contents of tube No. 1 are added to the contents of tube No. 2, incubated for 5 min at room temperature. The resulting solution was added dropwise to the cells in a volume of 40 μl.

As a control, a culture of endothelial cells from the human umbilical vein HUVEC is used, transfected with the GDTT1.8NAS3 DNA vector that does not contain the insert of the target P4HA2 gene. The preparation of the control vector GDTT1.8NAS3 for transfection is carried out as described above.

Isolation of total RNA from transfected cells and PCR amplification is carried out as in Example 6. The beta-2-microglobulin B2M gene is used as the reference gene. As a negative control, deionized water is used. The number of PCR products - cDNA of genes P4NA2 and B2M obtained as a result of amplification, is estimated in real time using the software of the Bio-RadCFXManager 2.1 amplifier.

In order to confirm the increase in the expression of the P4NA2 gene in the culture of endothelial cells from the human umbilical vein HUVEC (ATCC ^® CRL-1730 ™) after transfection of these cells with the genetic construct GDTT1.8NAS3-P4HA2 P4HA2 SEQ ID No: 4 in FIG. 22 shows the graphs of the accumulation of PCR products.

It follows from the figure that as a result of transfection of a culture of endothelial cells from the human umbilical vein HUVEC with the genetic construct GDTT1.8NAS3-P4HA2 P4NA2 SEQ ID No: 4, the level of specific mRNA of the human P4NA2 gene increased many times.

Example 15

The introduction into the human skin of a genetic construct with the cDNA of the P4NA2 gene in order to confirm the increase in the amount of protein shed 4-hydroxylase alpha 2 in human skin.

For the purpose of analyzing the increase in the quantitative level of protein, the patient was introduced the pCMV6-XL5 P4NA2 genetic construct SEQ ID No: 5 with cDNA of the P4HA2 gene (zone B) and a placebo was introduced, which was a combination of a vector plasmid containing no P4NA2 cDNA gene with a transport molecule (zone A) - into the skin of the forearm.

Transfection reagent cGMP grade in-vivo-jetPEI (Polyplus Transfection, France - USA) was used as a transport system. The genetic construct pCMV6-XL5 P4HA2 SEQ ID No: 5, which contains the modified cDNA of the P4NA2 gene (SEQ ID No: 5) and the plasmid pCMV6-XL5, used as a placebo - which does not contain the cDNA of the P4NA2 gene, each of which was dissolved in sterile water Nuclease-Free degree of purification. To obtain the genetic construct, DNA-cGMP grade in-vivo-jetPEI complexes were prepared in accordance with the manufacturer's recommendations.

The calculation of the amount of reagent was carried out according to the formula proposed by the manufacturer:

V (μl) = (pDNA (μg) × 3 × N / P) / 150

where N / P is the ratio between the number of nitrogenous residues in jetPEI and the number of phosphate groups of nucleic acids. In this experiment, the ratio N / P = 6 was used, i.e., for 20 μg pDNA - 2.4 μl jetPEI.

The resulting genetic construct and placebo were used for administration to the patient. The introduction was carried out by the tunnel method with a 30G needle to a depth of 3 mm. The volume of the injected solution of the genetic construct and placebo is about 0.3 ml for each. The foci of the introduction of the genetic construct and placebo were located at a distance of 3-5 cm from each other.

The amount of protein shed by 4-hydroxylase alpha 2 was evaluated in lysates of biopsy samples of the patient’s skin according to the method described in Example 8.

Biopsy samples were taken 4 days after the introduction of the genetic construct. A biopsy was performed from skin areas in the area of introduction of the genetic construct and placebo, as well as from intact skin, using a biopsy device Epitheasy 3.5 (Medax SRL) further, as described in example 9.

It was shown that in the patient’s skin in the area of the introduction of the pCMV6-XL5 P4NA2 genetic construct SEQ ID No: 5 with the cDNA of the P4NA2 gene, there was an increase in the amount of protein shed by 4-hydroxylase alpha 2, while with placebo, the amount of protein shed by 4-hydroxylase alpha 2 in the skin did not change. The result indicates enhanced expression of the P4NA2 gene when using the pCMV6-XL5 P4NA2 genetic construct SEQ ID No: 5 with cDNA of the P4NA2 gene and, accordingly, the effectiveness of this genetic construct. The results are shown in figure 23.

Example 16

The introduction of the genetic construct with the cDNA of the P4NA2 gene into human cartilage tissue with the aim of confirming an increase in the amount of protein shed 4-hydroxylase alpha 2 in human cartilage tissue.

In order to analyze the change in the amount of protein, 4-hydroxylase alpha 2 was shed, 2 patients were injected with the VTvaf17 P4NA2 genetic construct SEQ ID No: 6 with cDNA of the P4NA2 gene without the transport system (zone B) and a placebo was introduced, which was a combination of the vector plasmid VTvaf17 without the cDNA of the P4NA2 gene without transport system (zone A) - into the cartilaginous tissue of the auricles on the back side.

The genetic design of VTvaf17 P4NA2 SEQ ID No: 6, with cDNA of the P4HA2 gene (SEQ ID No: 6), and plasmid VTvaf17, used as a placebo, which does not contain cDNA of the P4NA2 gene, was dissolved in sterile water of the Nuclease-Free purification grade.

The resulting genetic construct and placebo were used for administration to the patient. The introduction was carried out by the tunnel method with a 30G needle to a depth of 1.5-2 mm into the cartilaginous tissue of the auricles from the back side. The volume of the injected solution of the genetic construct and placebo is about 0.2 ml for each, the concentration of the genetic construct is 0.5 mg / ml. The foci of introduction of the genetic construct and placebo were located at a distance of 2-3 cm from each other.

The amount of protein shed by 4-hydroxylase alpha 2 was evaluated in the lysates of the biopsy samples of the cartilage of the patient according to the method described in Example 8.

Biopsy samples were taken 3 days after the introduction of the genetic construct. The biopsy was carried out from the cartilage tissue sites in the area of introduction of the genetic construct, as well as from the intact sites and in the area of the placebo injection, by percutaneous puncture biopsy using a disposable manual biopsy needle, then according to Example 9.

It was shown that in the patient’s cartilaginous tissue in the region of the introduction of the VTvaf17 P4NA2 genetic construct SEQ ID No: 6 with the cDNA of the P4NA2 gene, there was an increase in the amount of protein shedding 4-hydroxylase alpha 2, whereas with placebo, the amount of protein shedding 4-hydroxylase alpha 2 in the cartilage tissue did not change. The result indicates enhanced expression of the P4NA2 gene when using the VTvaf17 P4NA2 genetic construct SEQ ID No: 6 and, accordingly, the effectiveness of this construct. The results are shown in figure 24

Example 17

The introduction of the genetic construct with the cDNA of the P4NA2 gene into human muscle tissue in order to confirm an increase in the amount of protein shed 4-hydroxylase alpha 2 in human muscle tissue.

In order to analyze the change in the amount of protein, the patient was introduced the GDTT1.8NAS1-P4HA2 P4HA2 SEQ ID No: 7 construct with cDNA of the P4NA2 gene (zone B) and a placebo, which is a combination of the vector plasmid GDTT1.8NAS1 without the cDNA of the P4NA2 gene with the transport molecule, was introduced (zone A) - into muscle tissue in the area of the upper third of the forearm.

Polyethyleneimine transfection reagent cGMP grade in-vivo-jetPEI (Polyplus Transfection, France - USA) was used as a transport system.

The genetic construct GDTT1.8NAS1-P4HA2 P4HA2 SEQ ID No: 7, with the cDNA of the P4HA2 gene (SEQ ID No: 7), and the plasmid GDTT1.8NAS1, used as a placebo, which does not contain the cDNA of the P4NA2 gene, was dissolved in sterile water of the degree of purification Nuclease-Free. To obtain the genetic construct, DNA-cGMP grade in-vivo-jetPEI complexes were prepared in accordance with the manufacturer's recommendations.

The calculation of the amount of reagent was carried out according to the formula proposed by the manufacturer:

V (μl) = (pDNA (μg) × 3 × N / P) / 150

where N / P is the ratio between the number of nitrogenous residues in jetPEI and the number of phosphate groups of nucleic acids.

The resulting genetic construct and placebo were used for administration to the patient. The introduction was carried out by the tunnel method with a 30G needle to a depth of 15-20 mm. The volume of the injected solution of the genetic construct and placebo is about 0.5 ml for each. The foci of the introduction of the genetic construct and placebo were located at a distance of 5-7 cm from each other

The amount of protein shed by 4-hydroxylase alpha 2 was measured in lysates of biopsy samples of muscle tissue of the patient according to the method described in Example 8.

Biopsy samples were taken 3 days after the introduction of the genetic construct. A biopsy was performed from muscle tissue in the area where the genetic construct was introduced, as well as from intact areas of the muscle and in the placebo using the MAGNUM automatic biopsy device (BARD, USA) in a volume of at least 25 cubic mm, then follow the example 9.

It was shown that in the patient’s muscle tissue in the area of the introduction of the genetic construct GDTT1.8NAS1-P4HA2 P4NA2 SEQ ID No: 7 there was an increase in the amount of protein shedding 4-hydroxylase alpha 2, whereas when placebo was introduced, the amount of protein shed 4-hydroxylase alpha 2 in muscle tissue did not change. The result indicates enhanced expression of the P4NA2 gene using the GDTT1.8NAS1-P4HA2 P4HA2 SEQ ID No: 7 genetic construct and, accordingly, the effectiveness of this genetic construct. The results are shown in figure 25.

Example 18

The introduction to a group of different patients of genetic constructs containing modified cDNA of the P4NA2 gene and a portion of the native unmodified cDNA of the P4NA2 gene.

In order to confirm the individual nature of the increase in the expression level of the target P4NA2 gene to a different level when genetic constructs with modified and unmodified cDNA of the P4NA2 gene were introduced into the skin of the patients, the level of protein shed of 4-hydroxylase alpha 2 was analyzed in the skin biopsy sample of the patient group depending on the presence and types of modifications in the cDNA of the P4NA2 gene.

The sequence of the portion of the native unmodified cDNA of the P4NA2 gene is shown in Figure 1, SEQ P4HA2 ID No: 1, the sequences of the modified cDNA of the P4HA2 gene are shown in Figures 2-7 (SEQ P4HA2 ID No: 2, SEQ P4HA2 ID No: 3, SEQ P4HA2 ID No: 4, SEQ P4HA2 ID No: 5, SEQ P4HA2 ID No: 6, SEQ P4HA2 ID No: 7).

Moreover, the genetic constructs based on VTvaf17, one of which contains a portion of the native unmodified cDNA of the P4NA2 gene (SEQ ID No: 1), the second genetic construct contains a portion of the modified cDNA of the P4NA2 gene (SEQ ID No: 2), the third genetic construct contains the modified cDNA of the gene P4NA2 (SEQ ID No: 3), the fourth genetic construct contains a modified cDNA of the P4NA2 gene (SEQ ID No: 4), the fifth genetic construct contains a modified cDNA of the P4NA2 gene (SEQ ID No: 5), the sixth genetic construct contains a modified cDNA gene P4NA2 (SEQ ID No: 6), the seventh genetic construct contains a modified cDNA of the gene P4NA2 (SEQ ID No: 7), the eighth genetic construct (placebo), is a VTvaf17 vector, was dissolved in sterile water with Nuclease-Free purification. DNA constructs were prepared by DNA complexes in combination with a transport molecule - Transfection reagent cGMP grade in-vivo-jetPEI (Polyplus Transfection, France - USA) in Example 15.

The resulting seven variants of genetic constructs and a placebo were used for administration to the skin of patients. The introduction was carried out by the tunnel method with a 30G needle to a depth of 3 mm. The volume of the injected solution of each genetic construct was about 0.3 ml. The foci of the introduction of genetic constructs and placebo were located at a distance of 3-5 cm from each other.

Each of the 32 patients who were randomly selected was injected with 7 genetic constructs and a placebo into the skin of the forearm.

Biopsy samples were taken 72 hours after the introduction of genetic constructs. The biopsy was performed from the sites of the introduction of genetic constructs, placebo, as well as from intact skin using a device for taking a skin biopsy Epitheasy 3.5 (Medax SRL). The patient’s skin was pre-washed with sterile saline and anesthetized with lidocaine solution. The volume of each biopsy sample is at least 10 cubic meters. mm, mass - not less than 11 mg. The sample was placed in a buffer solution containing 50 mM Tris-HCI pH 7.6, 100 mM NaCl, 1 mM EDTA and 1 mM phenylmethylsulfonyl fluoride and homogenized to obtain a uniform suspension. The resulting suspension was centrifuged for 10 minutes at 14,000 rpm. The supernatant was taken and used to quantify the target protein.

A - biopsy obtained after the introduction of a genetic construct based on VTvaf17 P4NA2 SEQ ID No: 1, containing unmodified cDNA of the P4NA2 gene (SEQ ID No: 1.) according to Example 2 in combination with a transport molecule - Transfection reagent cGMP grade in-vivo-jetPEI (Polyplus Transfection, France - USA) no. Example 15.

C - biopsy obtained after the introduction of a genetic construct based on VTvaf17 P4NA2 SEQ ID No: 2, containing the modified cDNA of the P4NA2 gene (SEQ ID No: 2.) according to Example 3 in combination with a transport molecule - Transfection reagent cGMP grade in-vivo-jetPEI (Polyplus Transfection, France - USA) according to example 15.

C - biopsy obtained after the introduction of a genetic construct based on VTvaf17 P4NA2 SEQ ID No: 3, containing the modified cDNA of the P4NA2 gene (SEQ ID No: 3.) according to Example 3 in combination with a transport molecule - Transfection reagent cGMP grade in-vivo-jetPEI (Polyplus Transfection, France-USA) according to example 15.

D - biopsy obtained after the introduction of a genetic construct based on VTvaf17 P4NA2 SEQ ID No: 4, containing the modified cDNA of the P4NA2 gene (SEQ ID No: 4.) according to Example 3 in combination with a transport molecule - Transfection reagent cGMP grade in-vivo-jetPEI (Polyplus Transfection, France - USA) according to example 15.

E - biopsy obtained after the introduction of a genetic construct based on VTvaf17 P4NA2 SEQ ID No: 5, containing the modified cDNA of the P4NA2 gene (SEQ ID No: 5) according to Example 3 in combination with a transport molecule - Transfection reagent cGMP grade in-vivo-jetPEI ( Polyplus Transfection, France - USA) as in Example 15.

F - biopsy obtained after the introduction of a genetic construct based on VTvaf17 P4NA2 SEQ ID No: 6, containing the modified cDNA of the P4NA2 gene (SEQ ID No: 6) according to Example 3 in combination with a transport molecule - Transfection reagent cGMP grade in-vivo-jetPEI ( Polyplus Transfection, France - USA) as in Example 15.

G - biopsy obtained after the introduction of the genetic construct based on VTvaf17 P4NA2 SEQ ID No: 7, containing the modified cDNA of the P4NA2 gene (SEQ ID No: 7.) according to Example 3 in combination with a transport molecule - Transfection reagent cGMP grade in-vivo-jetPEI (Polyplus Transfection, France - USA) according to example 15.

H is the biopsy obtained after the introduction of the vector plasmid VTvaf17 that does not contain the cDNA of the P4NA2 gene according to Example 3 in combination with the transport molecule Transfection reagent cGMP grade in-vivo-jetPEI (Polyplus Transfection, France-USA) according to Example 15.

The amount of protein shedding 4-hydroxylase alpha 2 was determined in nine skin biopsies from each patient (A to H and intact skin biopsies) 72 hours after the introduction of genetic constructs by enzyme-linked immunosorbent assay (ELISA) using the Human P4NA2 ELISA Kit ( MyBioSource, USA), as described in Example 8.

Based on the results of the quantitative analysis of the protein, he shed 4-hydroxylase alpha 2, we selected indicators for each biopsy from each patient, which showed the maximum levels of the amount of protein shed 4-hydroxylase alpha 2 and combined them into groups based on the following criterion:

In group 1, the maximum amount of protein shed 4-hydroxylase alpha 2, was observed with the introduction of unmodified cDNA of the P4NA2 gene SEQ ID No: 1. This group included 7 biopsy samples out of 32.

In group 2, the maximum amount of protein shed 4-hydroxylase alpha 2 was observed with the introduction of the modified cDNA of the P4NA2 gene SEQ ID No: 2. This group included 6 of 32 biopsy samples.

In group 3, the maximum amount of protein shed 4-hydroxylase alpha 2, was observed with the introduction of the modified cDNA of the P4NA2 gene SEQ ID No: 3. This group included 4 biopsy samples from 32.

In group 4, the maximum amount of protein shed 4-hydroxylase alpha 2 was observed with the introduction of the modified cDNA of the P4NA2 gene SEQ ID No: 4. This group included 4 biopsy samples from 32.

In group 5, the maximum amount of protein shed 4-hydroxylase alpha 2, was observed with the introduction of a modified cDNA of the P4NA2 gene SEQ ID No: 5. This group included 3 biopsy samples from 32

In group 6, the maximum amount of protein shed 4-hydroxylase alpha 2 was observed with the introduction of the modified cDNA of the P4NA2 gene SEQ ID No: 6. This group included 4 biopsy samples from 32.

In group 7, the maximum amount of protein shed 4-hydroxylase alpha 2 was observed with the introduction of the modified cDNA of the P4NA2 gene SEQ ID No: 7. This group included 4 biopsy samples from 32.

None of the biopsy specimens that received placebo as a vector plasmid containing the P4NA2 gene cDNA contained the maximum amount of protein shedding 4-hydroxylase alpha 2, unlike the biopsy specimens that were introduced into the genetic constructs containing the cDNA gene P4NA2.

Figure 26 shows diagrams of protein concentration indicators (averaged within the group if more than one biopsy is included in the group) for each group of biopsy samples for all genetic constructs participating in the experiment after the introduction of the genetic constructs containing modified and native cDNA of the P4NA2 gene.

From this example, it follows that the achievement of the maximum level of protein shed 4-hydroxylase alpha 2 in biopsies of the skin of various patients with the introduction of genetic constructs into the skin is associated with individual characteristics of the patients and depends on the presence and type of modifications in the cDNA of the P4NA2 gene that are part of the genetic designs.

Each genetic construct from the group of genetic constructs is effective in some significant group of patients. Therefore, to select the most effective genetic construct from the group of genetic constructs for therapeutic purposes, a preliminary personalized study of the biopsy of the patient, or cells grown from these biopsy specimens for maximum therapeutic effect, created by the genetic constructs within the group of genetic constructs, is necessary.

Example 19

Transfection of a patient’s fibroblast cell line with different genetic constructs containing modified P4NA2 gene cDNA and a portion of a native unmodified P4NA2 gene cDNA to personalize the most effective genetic construct for a given patient for subsequent transfection with the patient’s genetic construct or to introduce this genetic construct to the patient as part of a therapeutic procedure.

In order to determine the most effective genetic construct for a particular patient, the amount of protein prolyl 4-hydroxylase alpha 2 was analyzed in the patient’s cell fibroblast lysates transfected with different genetic constructs containing a portion of the native unmodified cDNA of the P4NA2 gene and modified cDNA of the P4NA2 gene.

The sequence of the portion of the native unmodified cDNA of the P4NA2 gene is shown in Figure 1, SEQ P4HA2 ID No: 1, the sequences of the modified cDNA of the P4HA2 gene are shown in Figures 2-7 (SEQ P4HA2 ID No: 2, SEQ P4HA2 ID No: 3, SEQ P4HA2 ID No: 4, SEQ P4HA2 ID No: 5, SEQ P4HA2 ID No: 6, SEQ P4HA2 ID No: 7).

Fibroblast cultures from the patient’s biopsy were grown according to Example 5, aliquots were taken and cell lysis was performed: the cell pellet corresponding to 1 × 10 ⁶ cells was washed with phosphate buffer and resuspended in ice in lysis buffer containing 25 mM HEPES pH 7.9, 100 mM NaCl, 1 mM EDTA, 1% Triton X-100, 10% glycerol and 1 mM phenylmethylsulfonyl fluoride. The lysate was centrifuged for 5 minutes at 14,000 rpm.

Then the patient’s fibroblast culture was divided into 27 parts. One part, designated (A), was transfected according to Example 7 with the pCMV6-XL5 genetic construct SEQ ID No: 1 containing unmodified cDNA of the P4NA2 gene (SEQ ID No: 1.) according to Example 2 in combination with the transport liposome molecules of Example 9.

The second part, designated (B), was transfected according to Example 7 with the pCMV6-XL5 genetic construct SEQ ID No: 2 containing a variant of the modified cDNA of the P4NA2 gene (SEQ ID No: 2.) according to Example 3 in combination with the transport lipid molecules of Example 9 .

The 3rd part, designated (C), was transfected according to Example 7 with the pCMV6-XL5 genetic construct SEQ ID No: 3 containing a variant of the modified cDNA of the P4NA2 gene (SEQ ID No: 3.) according to Example 3 in combination with transport molecules - liposomes according to example 9.

The 4th part, designated (D), was transfected according to Example 7 with the pCMV6-XL5 genetic construct SEQ ID No: 4 containing a variant of the modified cDNA of the P4NA2 gene (SEQ ID No: 4.) according to Example 3 in combination with transport molecules - liposomes according to example 9.

The 5th part, designated (E), was transfected according to Example 7 with the pCMV6-XL5 genetic construct SEQ ID No: 5 containing a variant of the modified cDNA of the P4NA2 gene (SEQ ID No: 5.) according to Example 3 in combination with transport molecules - liposomes according to example 9.

The 6th part, labeled (F), was transfected according to Example 7 with the pCMV6-XL5 genetic construct SEQ ID No: 6 containing a variant of the modified P4NA2 gene cDNA (SEQ ID No: 6) according to Example 3 in combination with the transport molecules - liposomes according to Example 9.

The 7th part, designated (G), was transfected according to Example 7 with the pCMV6-XL5 genetic construct SEQ ID No: 7 containing a variant of the modified cDNA of the P4NA2 gene (SEQ ID No: 7) according to the example in combination with the transport molecules - liposomes according to Example 9 .

The 8th part, designated (H), was transfected according to Example 7 with the vector plasmid pCMV6-XL5 that did not contain the cDNA of the P4NA2 gene in combination with the transport molecules - liposomes of Example 9.

Determination of the amount of protein shed by 4-hydroxylase alpha 2 in the patient’s cell fibroblast lysates was performed 72 hours after transfection according to the method described in Example 8.

According to the results of determining the amount of protein, 4-hydroxylase alpha 2 was shed, a variant of the genetic construct was isolated in the patient’s fibroblast culture, during transfection of which the maximum amount of protein shed 4-hydroxylase alpha 2 was observed in the cell culture lysate, which ultimately indicates the maximum expression of the P4NA2 gene. In this experiment, the maximum amount of protein in the lysate was observed during transfection with the pCMV6-XL5 P4NA2 genetic construct SEQ ID No: 4 containing the modified cDNA of the P4NA2 gene, as shown in Figure 27.

Thus, the most effective genetic construct was applied to this patient for subsequent transfection of the patient's cells as part of a therapeutic procedure.

Example 20

The introduction into the patient’s skin of various genetic constructs containing modified cDNA of the P4NA2 gene or a portion of the native unmodified cDNA of the P4NA2 gene to select from the group the most effective genetic construct for this patient for subsequent administration of this construct to the patient as part of a therapeutic procedure.

In order to determine the most effective genetic construct for a particular patient, the amount of protein in the supernatant of skin biopsy samples of this patient was analyzed after the introduction of genetic constructs containing modified P4NA2 cDNA or a portion of the native P4NA2 gene cDNA into his skin.

The sequence of the portion of the native unmodified cDNA of the P4NA2 gene is shown in Figure 1, SEQ P4HA2 ID No: 1, the sequences of the modified cDNA of the P4HA2 gene are shown in Figures 2-7 (SEQ P4HA2 ID No: 2, SEQ P4HA2 ID No: 3, SEQ P4HA2 ID No: 4, SEQ P4HA2 ID No: 5, SEQ P4HA2 ID No: 6, SEQ P4HA2 ID No: 7).

The patient was given 7 genetic constructs and a placebo into the skin of the forearm.

1st genetic construct (A) pCMV6-XL5 SEQ ID No: 1, containing a portion of the native unmodified cDNA of the P4NA2 gene (SEQ ID No: 1.) according to Example 2 in combination with a transport molecule - Transfection reagent cGMP grade in vivo-jetPEI (Polyplus Transfection, France - USA) according to example 15.

2nd genetic construct (B) pCMV6-XL5 SEQ ID No: 2, containing a portion of the modified cDNA of the P4NA2 gene (SEQ ID No: 2.) according to Example 3 in combination with a transport molecule - Transfection reagent cGMP grade in-vivo-jetPEI ( Polyplus Transfection, France-USA) according to example 15.

3rd genetic construct (C) pCMV6-XL5 SEQ ID No: 3, containing the modified cDNA region of the P4NA2 gene (SEQ ID No: 3.) according to example 3 in combination with a transport molecule - Transfection reagent cGMP grade in-vivo-jetPEI ( Polyplus Transfection, France - USA) as in Example 15.

4th genetic construct (D) pCMV6-XL5 SEQ ID No: 4, containing the modified P4NA2 gene cDNA region (SEQ ID No: 4.) according to Example 3 in combination with a transport molecule - Transfection reagent cGMP grade in-vivo-jetPEI ( Polyplus Transfection, France-USA) according to example 15.

5th genetic construct (E) pCMV6-XL5 SEQ ID No: 5 containing a section of the modified cDNA of the P4NA2 gene (SEQ ID No: 5.) according to Example 3 in combination with a transport molecule - Transfection reagent cGMP grade in-vivo-jetPEI ( Polyplus Transfection, France-USA) according to example 15.

6th genetic construct (F) pCMV6-XL5 SEQ ID No: 6, containing the modified P4NA2 gene cDNA region (SEQ ID No: 6.) according to Example 3 in combination with a transport molecule - Transfection reagent cGMP grade in-vivo-jetPEI ( Polyplus Transfection, France-USA) according to example 15.

7th genetic construct (G) pCMV6-XL5 SEQ ID No: 7, containing the modified P4NA2 gene cDNA region (SEQ ID No: 7.) according to Example 3 in combination with the transport molecule -Transfection reagent cGMP grade in-vivo-jetPEI ( Polyplus Transfection, France-USA) according to example 15.

The 8th vector plasmid pCMV6-XL5 (H) not containing the cDNA of the P4NA2 gene according to Example 3 in combination with the transport molecule Transfection reagent cGMP grade in-vivo-jetPEI (Polyplus Transfection, France-USA) according to Example 15.

Moreover, the genetic constructs, one of which contains a portion of the native unmodified cDNA of the P4NA2 gene (SEQ ID No: 1), the second genetic construct contains the modified cDNA of the P4NA2 gene (SEQ ID No: 2), and the third genetic construct contains the modified cDNA of the P4NA2 gene (SEQ ID No: 3), the fourth genetic construct contains the modified cDNA of the P4NA2 gene (SEQ ID No: 4), the fifth genetic construct contains the modified cDNA of the P4NA2 gene (SEQ ID No: 5), the sixth genetic construct contains the modified cDNA of the P4NA2 gene (SEQ ID No: 6), s dmaya genetic construct comprises a cDNA R4NA2 modified gene (SEQ ID No: 7), eighth plasmid (placebo) is a vector pCMV6 XL5, dissolved in sterile water Nuclease-Free degree of purification. To obtain the genetic construct, DNA-polyethyleneimine Transfection reagent cGMP grade in-vivo-jetPEI (Polyplus Transfection, France-USA) complexes were prepared according to Example 15.

The resulting seven variants of genetic constructs and a placebo were used for administration to a patient. The introduction was carried out by the tunnel method with a 30G needle to a depth of 3 mm. The volume of the injected solution of each genetic construct was about 0.2 ml. The skin at the injection site was labeled, foci of introduction of genetic constructs and placebo were located at a distance of 3-4 cm from each other.

Biopsy samples were taken 72 hours after the introduction of genetic constructs. The biopsy was performed from the sites of the introduction of genetic constructs, placebo, as well as from intact skin using a device for taking a skin biopsy Epitheasy 3.5 (Medax SRL). The patient’s skin was pre-washed with sterile saline and anesthetized with lidocaine solution. The size of each biopsy sample was at least 10 cubic meters. mm, mass - not less than 11 mg.

Each sample was placed in a buffer solution containing 50 mM Tris-HCl pH 7.6, 100 mM NaCl, 1 mM EDTA and 1 mM phenylmethyl sulfonyl fluoride and homogenized to obtain a uniform suspension. The resulting suspension was centrifuged for 10 minutes at 14,000 rpm. The supernatant was taken and used to quantify the target protein.

Determination of the amount of protein shed by 4-hydroxylase alpha 2 was carried out in nine biopsies of the patient’s skin 72 hours after the introduction of the genetic constructs according to the method described in Example 8.

According to the results of determining the amount of protein, 4-hydroxylase alpha 2 was shed, a variant of the genetic construct was isolated in the supernatant of the biopsy samples of the patient’s skin, with the introduction of which the maximum amount of protein in the biopsy specimen was observed, which ultimately indicates the maximum expression of the P4NA2 gene. In this experiment, the maximum amount of the target protein was shed by 4-hydroxylase alpha 2 in the supernatant of the skin biopsy specimen was noted with the introduction of the genetic construct pCMV6-XL5 P4NA2 SEQ ID No: 1, containing the native cDNA of the P4NA2 gene, as shown in Figure 28.

Thus, the most effective genetic construct for this patient was chosen. A genetic construct was created on the basis of a non-viral vector plasmid including the cDNA of the P4NA2 gene to stop the manifestations of the human body conditions associated with a decrease in the expression of the P4NA2 gene and / or a decrease in the amount of protein shed by 4-hydroxylase alpha 2, method for its preparation and use.

The created genetic construct for stopping the manifestations of human body conditions associated with a decrease in the expression of the P4NA2 gene and / or a decrease in the amount of protein shed by 4-hydroxylase alpha 2, is selected from the group of genetic constructs, each of which is based on a vector non-viral plasmid that includes a native or modified cDNA the P4NA2 gene, with the coding sequence of the protein, shed 4-hydroxylase alpha 2.

The created genetic construct based on a non-viral DNA vector including P4NA2 cDNA for stopping the manifestations of human body conditions associated with a decrease in P4NA2 gene expression and / or a decrease in the amount of protein shed 4-hydroxylase alpha 2, selected from the group of genetic constructs with one of the modified cDNA of the P4NA2 gene or with a portion of the native unmodified cDNA of the P4HA2 gene, allows in practice to increase the expression of the protein shed by 4-hydroxylase alpha 2 to the required level in various cells organs and tissues and / or organs and tissues of a person by increasing the expression of the P4NA2 gene.

As a result of preliminary studies of the biopsy of the patient, or cells grown from these biopsies, determine which version of the genetic construct from the created group of genetic constructs must be selected for this patient in order to increase the amount of protein shed by 4-hydroxylase alpha 2 in the cells of these organs and tissues and / or organs and tissues to the required level for a particular patient. In cases where the use of a genetic construct with a portion of the native unmodified cDNA of the P4NA2 gene does not lead to the desired change in the quantitative level of the protein shed by 4-hydroxylase alpha 2 in the cells of organs and tissues and / or organs and tissues, it is necessary to apply a genetic construct with a modified cDNA of the gene P4NA2 leading to more efficient expression of the P4NA2 gene.

A genetic construct selected from the group of genetic constructs with one of the modified cDNA of the P4NA2 gene or with a portion of the native unmodified cDNA of the P4NA2 gene provides a high level of expression of the P4NA2 gene by increasing the amount of protein shed by 4-hydroxylase alpha 2 in the cells of organs and tissues and / or organs and human tissues, namely, in hematopoietic cells, or mesenchymal stem cells, or chondroblasts, or myocytes, or myoblasts, or fibroblasts, or osteoblasts, or keratocytes, or epithelial cells, or corneal cells, or endothelial cells, or epithelial cells, in combination with or without a transport molecule when transfected with these genetic constructs of cells of human organs and tissues and / or in human organs and tissues, namely, in connective tissue structures, or skin, or joints, or cartilage, or bone, or muscle, or tendons, or ligaments, or blood vessels, or the wall of arteries, or the cornea of the eye, or the sclera, or placenta, or dentin, or stroma of internal organs in combination transport molecule with or without the introduction of genetic constructs into human organs and tissues.

Thus, the above examples confirm the fulfillment of the task, namely, the creation of a genetic construct based on a non-viral vector plasmid, including cDNA of the P4NA2 gene, to stop the manifestations of the human body conditions associated with a decrease in the expression of the P4NA2 gene and / or a decrease in the amount of protein shed by 4-hydroxylase alpha 2, selected from the group of genetic constructs, each of which is created on the basis of a vector non-viral plasmid, including native or modified cDNA of the P4NA2 gene, using which, taking into account the individual characteristics of the patient, there is an increase in the level of expression of the P4NA2 gene and / or an increase in the amount of protein shed by 4-hydroxylase alpha 2 in the cells of organs and tissues and / or organs and tissues of the body.

In addition, with the introduction of genetic constructs, in contrast to the introduction of the protein itself, the required frequency of their introduction is reduced due to the prolonged action, and intracellular delivery is also facilitated.

When using the claimed genetic design selected from the group, the exogenous genetic material is not embedded in the cell genome. In the case of using a genetic construct based on the vectors VTvaf17 and GDTT1.8NAS1, GDTT1.8NAS3, GDTT1.8NAS7, the probability of embedding viral genome elements in the patient’s genome and the occurrence of antibiotic resistance in the patient due to the performed therapeutic procedure is absolutely excluded.

An increase in the efficiency of the correction of the quantitative level of a protein shed by 4-hydroxylase alpha 2 in the cells of human organs and tissues is achieved due to the fact that with an insufficient amount of this protein due to a defect or insufficient expression of the P4NA2 gene, not a protein is used, but a genetic construct with cDNA of the P4NA2 gene encoding protein.

Industrial applicability.

All the above examples on the creation and use of the created genetic constructs selected from the group of genetic constructs confirm its industrial applicability.

List of abbreviations

DNA - deoxyribonucleic acid

cDNA - complementary deoxyribonucleic acid

RNA - ribonucleic acid

mRNA - template ribonucleic acid

PCR - polymerase chain reaction

ml - milliliter

μl - microliter

l - liter

cube mm - cubic millimeter

mcg - micrograms

mg - milligram

g - gram

μM - micromol

mm - millimole

rpm - revolutions per minute

nm - nanometer

cm - centimeter

MW - milliwatts

father f-relative fluorescence unit

GK - genetic design

Claims (6)

1. A genetic construct based on a non-viral DNA vector, including cDNA of the P4NA2 gene, for stopping the manifestations of human body conditions associated with a decrease in the expression of the P4NA2 gene and / or a decrease in the amount of protein shed by 4-hydroxylase alpha 2, selected from genetic constructs, each of which represents a genetic construct based on a DNA vector including cDNA of the P4NA2 gene, with the coding sequence of the protein shed 4-hydroxylase alpha 2, with deletions of 5'- and 3'-untranslated regions, namely obtained based on a portion of the native unmodified cDNA of the P4NA2 gene of SEQ ID No: 1 or a modified cDNA of the P4NA2 gene, while SEQ ID No: 2, or SEQ ID No: 3, or SEQ ID No: 4, or SEQ ID No: 4, or SEQ ID No: 5, or SEQ ID No: 6, or SEQ ID No: 7, or a combination of these genetic constructs, each of which also contains regulatory elements that provide a high level of expression of the P4NA2 gene in eukaryotic cells, namely in cells of organs and human tissues, and capable of increasing the amount of protein shed 4-hydroxylase alpha 2 in organ cells ov and tissues, namely in hematopoietic cells, or mesenchymal stem cells, or chondroblasts, or myocytes, or myoblasts, or fibroblasts, or osteoblasts, or keratocytes, or epithelial cells, or corneal cells, or endothelial cells, or epithelial cells, combination with or without a transport molecule during transfection with these genetic constructs of cells of human organs and tissues, and / or in human organs and tissues, namely in connective tissue structures, or skin, or joints, or cartilaginous tissue ani, or bone tissue, or muscle tissue, or tendons, or ligaments, or blood vessels, or the wall of arteries, or the cornea of the eye, or sclera, or placenta, or dentin, or stroma of internal organs, in combination with or without a transport molecule with the introduction of these genetic structures into organs and tissues of a person. 2. The genetic construct according to claim 1, characterized in that each genetic construct selected from one or more genetic constructs with a modified cDNA of the P4NA2 gene contains a nucleotide sequence that includes a protein-coding region of the cDNA of the P4NA2 gene that carries modifications that do not affecting the structure of the protein shed 4-hydroxylase alpha 2 or affecting the structure of the protein shed 4-hydroxylase alpha 2 and affecting the amino acid sequence encoded by this sequence, namely the nucleotide other substitutions leading to amino acid substitutions or termination of the amino acid chain, and / or deletion, or a combination of the above modifications. 3. The genetic construct according to claim 1, characterized in that liposomes, or dendrimers of the 5th and higher generations, or amphiphilic block copolymers, or a polyethyleneimine-based reagent are used as a transport molecule. 4. The method of obtaining the genetic construct according to claim 1, which consists in obtaining each genetic construct selected from one or more genetic constructs according to claim 1, wherein cDNA of the P4NA2 gene is obtained, then cDNA is placed in a vector plasmid capable of providing a high level of expression of this cDNA in the cells of various organs and tissues of a person, build up and secrete the necessary amount of genetic constructs, then combine the genetic construct with a transport molecule to transfect the resulting genetic by constructing cells of organs and tissues and / or introducing the obtained genetic construct into human organs and tissues, using cDNA of the P4NA2 gene with the coding sequence of the protein shed 4-hydroxylase alpha 2, with deletions of 5'- and 3'-untranslated regions, namely, the resulting based on a portion of a native unmodified cDNA of the P4NA2 gene of SEQ ID No: 1 or a modified cDNA of the P4NA2 gene, while either SEQ ID No: 2 or SEQ ID No: 3 or SEQ ID No: 4, or SEQ ID No: 4, or SEQ ID No: 5, or SEQ ID No: 6, or SEQ ID No: 7, or a combination of these genes iCal designs. 5. The method of using the genetic construct according to claim 1 for stopping the manifestations of human body conditions associated with a decrease in the expression of the P4NA2 gene and / or a decrease in the amount of protein shed by 4-hydroxylase alpha 2, which consists in transfecting one or more genetic constructs according to claim 1, by introducing into the patient’s organs and tissues autologous patient cells transfected with one or more genetic constructs according to claim 1, and / or introducing one or more genetic constants into the patient’s organs and tissues uktsy according to claim 1, or in a combination of ways indicated. 6. The method of applying the genetic construct according to claim 5, which consists in the fact that the choice of the genetic construct from the genetic constructs created according to claim 1 is made individually for each patient after a preliminary determination of the most effective genetic construct for this patient has been made.

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