RU2557385C1 - Codon-optimised sequences and pharmaceutical composition for recovery of blood vessels - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: codon-optimised cDNA are declared, encoding the stromal cell factor 1 alpha and the vascular endothelial growth factor of isoform 165, and also a recombinant plasmid containing them. The recombinant plasmid can be used as part of a pharmaceutical composition for the recovery of blood vessels and improvement of blood supply in the damaged tissues or organs.

EFFECT: invention enables to increase the efficiency of use of gene products.

4 cl, 5 dwg

Description

The invention relates to biotechnology, namely to genetic engineering and can be used in the creation of genetic therapeutic agents.

A genetic construct has been developed that allows simultaneous expression of several recombinant genes, which can be used in the development of drugs. A recombinant plasmid was created on the basis of the pBud (Kan) genetic construct, which includes codon-optimized nucleotide sequences of the genes of vascular endothelial growth factor isoform 165 (Eng.Vascular endothelial growth factor 165, VEGF165) and Stromal cell-derived factor 1α -1, SDF1-α),

Vascular endothelial growth factor VEGF-A, first described as vascular permeability factor (VPF), is the main regulator of angiogenesis and vasculogenesis. VEGF-A is a 34-42 kDa dimeric disulfide-linked glycoprotein. VEGF-A is a specific mitogen for vascular endothelial cells. VEGF-A induces the proliferation of endothelial cells (EC), their germination and the formation of capillary tubes. It is also a potent survival factor for EC and induces the expression of antiapoptotic proteins in these cells. Deletions of genes encoding VEGF-A lead to serious defects and abnormal development of the cardiovascular system.

The human VEGF-A gene is located at the 6p21.3 chromosomal locus. The coding region spans about 14 kb. VEGF-A exists in several isoforms: VEGF121, VEGF145, VEGF148, VEGF165, VEGF183, VEGF189, VEGF206, resulting from alternative splicing of mRNA, which consists of 8 exons. Each VEGF isoform corresponds to a specific extracellular localization based on their biochemical differences in the ability to bind heparin and heparan sulfate. So, all transcripts of the VEGF-A gene in humans contain exons 1-5 and 8, the differences are due to alternative splicing of exons 6 and 7.

VEGF165 is the isoform that is predominant in most tissues and is the most biologically active, it is the main protein that can bind heparin, and although a significant proportion of it remains bound to the cell, most are freely secreted. This isoform lacks amino acid residues, the incorporation of which into the protein molecule is encoded by exon 6. This isoform is the physiologically most common and most biologically active. VEGF165 is usually secreted as a 46 kDa homodimer, both subunits of which have a mass of 23 kDa.

Stromal cell factor 1 alpha (English Stromal cell-derived factor-1 alpha, SDF-1 alpha) is a small cytokine that belongs to the chemokine family, which is also called chemokine ligand 12 (Chemokine ligand 12, CXCL12 alpha). SDF-1 alpha and beta activate anti-inflammatory processes. CXCL12 causes lymphocyte chemotaxis.

CXCL12 is a ligand of the CXCR4 receptor. CXCR4-CXCL12 signaling induces chemotaxis, increased proliferation, and decreased apoptosis. During embryogenesis, it controls the migration of hematopoietic cells from the fetal liver to the bone marrow and the formation of large blood vessels. In adulthood, CXCL12 plays an important role in angiogenesis by attracting endothelial progenitor cells (EPCs) from the bone marrow through the CXC114-dependent mechanism.

An example of the application of the genetic engineering construct pBud (Kan) is the creation of a recombinant plasmid pBud (Kan) -coVEGF165-coSDF1-α .. For human diseases associated with impaired blood supply and damage to blood vessels, the formation of new elements of the vascular network due to the expression of VEGF165 and SDF1-α is able to restore blood supply to affected tissues and organs, as well as cause a stable positive effect. When using gene therapeutic drugs in vivo, only part of the cells is transfected with plasmid DNA. When using individual genetic constructs to express individual therapeutic genes, the chances that the cell will be transfected simultaneously with two different genetic constructs are significantly reduced.

The reason for combining precisely the vegf and sdf-1 genes in one plasmid was the establishment in the course of studies of the potentiating effect of the expression of these genes, which manifests itself not only in increasing the production of therapeutic proteins encoded by them, but also in the resulting biological effect. In addition, it was unexpectedly found that transfection of cells with a DNA plasmid with the vegf gene provides not only an increase in the production of the VEGF protein itself, but also SDF-1 protein (Fig. 1).

The principle of operation of the pBud (Kan) -coVEGF165-coSDF1-α genetic engineering construct is based on the fact that the drug is administered systemically or locally, followed by spontaneous transfection of resident cells, including endothelial cells, followed by expression of the vegf and sdf-1 genes. The expression of vegf and sdf-1 mRNAs is controlled by two strong eukaryotic promoters (immediate early cytomegalovirus CMV promoter and EF1 alpha promoter) and occurs independently. After processing of vegf and sdf-1 genes with mRNA, VEGF 165 and SDF1-α proteins are synthesized, which exhibit their normal biological activity as vascular endothelial growth factor and stromal cell factor.

Work on the use of VEGF 165 in therapeutic angiogenesis is widely carried out worldwide. One of the latest published works, Favaloro et al., Is given as an example of the clinical use of a plasmid containing VEGF 165 [1]. It is important that until today there have been no adverse or serious adverse events associated with the use of plasmids containing vascular endothelial growth factor, or complications after their use.

Moreover, at the current level of development of applied gene technologies, combining several genes encoding therapeutic proteins in a single gene construct is becoming an increasingly common approach. In particular, integration options such as the vegf-165 and bmp-7 genes in the adenovirus vector [2], etc. are known. Another option is a polycistronic plasmid DNA encoding four genes at once (oct4, sox2, klf4, and c-myc) shown for reprogramming somatic cells into induced pluripotent stem cells [3]. It should be noted that some authors have already mentioned the potential feasibility and feasibility of combining the sdf-1 and vegf genes in one expression vector for therapeutic indications [4] In the aforementioned publications, the researchers do not provide specific descriptions of the nucleotide sequences and do not even specify which variants of the sdf- genes 1 and vegf, they had in mind, citing only general hypothetical messages following from the modern level of technology. Moreover, it is the choice of specific variants of genes encoding strictly defined isoforms of therapeutic proteins, the correct optimization of the nucleotide sequences of transgenes, the development of an adequate expression vector that determines the achievement of the main technical result, which can only be the biological effectiveness of the developed gene-therapeutic construct, and not the feasibility of its creation.

The objective of the present invention is the creation of a plasmid genetic engineering construct, which allows you to simultaneously express several recombinant genes, exhibits high functional activity.

Brief Description of Drawings

Fig. 1. Dynamics of production of vegf and sdf-1α mRNA genes by MMSC culture transfected with a single vegf gene plasmid.

Fig. 2. Immunofluorescence analysis of VEGF 165 and SDF-1α expression in SH-SY5Y cells transfected with pBud (Kan) -VEGF 165-SDF-1α plasmid. 48 hours incubation after transfection. A, B, C, D, D, E, G, 3 — SH-SY5Y cells transfected with pBud (Kan) -VEGF 165-SDF-1α plasmid. And, K, L, M are non-transfected SH-SY5Y cells. A, D, I, - phase contrast light microscopy. B - staining using primary rabbit antibodies to stromal cell factor 1 and secondary donkey antibodies to rabbit immunoglobulin G conjugated with Alexa-488 fluorescent label. Ε - staining using primary rabbit antibodies to vascular endothelial growth factor and secondary donkey antibodies to rabbit immunoglobulin G conjugated with Alexa-488 fluorescent label. B, G, L — staining with DAPI fluorescent dye. G, Z, M - the combination of blue and green fluorescence spectra. Scale: 100 microns.

Fig. 3. Western blot analysis of the expression of VEGF 165 and SDF1-1α proteins in genetically modified HEK293T cells. Electrophoresis in 12% SDS-PAGE gel according to the Laemley system. Antibodies to VEGF (Santa cruz biotechnology; sc-507. Wells 1 and 2) and antibodies to SDF1 (Santa cruz biotechnology; sc-28876. Wells 3 and 4) were used at a 1:50 dilution. The expected protein size of VEGF 165 is 42 kDa, SDF-1α is 10 kDa. Wells 1, 3 are HEK293T cells transfected with the plasmid pBud (Kan) -VEGF165-SDF-1α. Wells 2, 4 are untransfected HEK293T cells. M - Prestained Protein Molecular Weight Marker (Thermo Fisher Scientific Inc.).

Fig. 4. The formation of capillary-like structures by HUVEC endothelial cells on Matrigel. Addition of culture medium at a final concentration of 30% collected from HEK293T cells transfected with plasmid pBud (Kan) -VEGF165-SDF-1α (A), B - culture medium of non-transfected cells (NTC) at a final concentration of 30%. Scale: 500 microns.

Fig. 5. Production of chemokines / cytokines IL-8 and MCP-1 by native (NTC) and genetically modified hADSCs cells.

To create the construct, the expression vector pBudCE4.1 (Invitrogen, Catalog # V532-20, USA) was used, which contains the CMV and EF1-α promoters for efficient transgene expression. We made changes to the nucleotide sequence of the plasmid vector pBudCE4.1:

1. The sequence of the zeocin resistance gene and its promoter are replaced by the sequence of the kanamycin resistance gene. The zeocin and kanamycin resistance genes are used both for selection of the plasmid vector in bacterial cells and for selection of eukaryotic cells transfected with the plasmid vector. The replacement of the zeocin resistance gene is due to the fact that its expression in eukaryotic cells (including human cells) leads to the appearance of a foreign protein (gene expression product), which can cause antibiotic resistance and constitute a potential allergen.

2. V5-His and myc-His tag sequences removed.

As an example of the implementation of the genetic engineering construct pBud (Kan), a recombinant plasmid pBud (Kan) -coVEGF165-coSDF1-α (SEQ ID NO: 1) was created, which is a modified expression vector plasmid pBudCE4.1 in which codon-optimized cDNAs were cloned, encoding stromal cell factor 1 alpha and vascular endothelial growth factor isoform 165 (at the restriction sites HindIII and XbaI), contains:

- CMV promoter 11-627 - Kozak sequences 695-698 and 3521-3524 - Gateway sites attBI and attBII 670-694 and 969-993 - CoSDF1-α gene 699-968 - Kanamycin resistance gene 1272-2313 - Promoter EF1-α 2329-3496 - HindIII and XbaI restriction sites 3515-3520 and 4101-4106 - gene coVEGF165 3525-4100

Codon optimization is based on the degeneracy of the genetic code, while the most common synonymic codons of the degenerate genetic code are used as optimal codons. The higher the frequency of one or another codon used to encode an amino acid in the body, the faster it will be translated by ribosomes due to the high intracellular concentration of tRNA recognizing such a donon. To optimize the codon composition of the vegf and sdf-1 genes, the OptimumGene algorithm was used, which takes into account various factors affecting gene expression levels, such as codon shift, GC composition, CpG dinucleotide content, secondary mRNA structure, tandem repeats, restriction sites, which may interfere with cloning, premature polyadenylation sites, additional minor ribosome binding sites. The nucleotide sequences of the mRNA of the vegf genes (GeneBank # AF486837.1, 576 bp) and sdf-1 (GeneBank # AY429472.1, 270 bp) were taken as a matrix for the base optimization.

The wild type of nucleotide sequences of the coding part of the vegf and sdf-1 genes contain a tandem of rare codons that can stop translation or reduce its efficiency. When optimizing the codon composition of the wild-type sdf-1 gene, the CAI codon adaptation index (Codon Adaptation Index) was improved from 0.77 to 0.88. When optimizing the wild-type codon composition of the VEGF 165 gene, the CAI codon adaptation index was improved from 0.81 to 0.87. To increase the stability of mRNA, the GC composition was optimized and extended regions with a high content of GC pairs were removed. In addition, the optimization process removed potential cis-acting sites. As a result of the bottom optimization, the amino acid sequences of the genes SDF1-A (SEQ ID N0: 2) and VEGF165 (SEQ ID NO: 3) did not change and amounted to 89 and 191 amino acid residues, respectively.

The de novo synthesis of the nucleotide sequence of the plasmid pBud (Kan) -coVEGF165-coSDFl-α, including vegf and sdf-1 genes optimized for codon composition, was carried out by GenScript (United States). Thus, the plasmid pBud (Kan) -coVEGF165-coSDF1-α encoding the codon-optimized sequences of the vegf and sdf-1 genes was obtained.

The functional activity of the genetic construct was confirmed by in vitro analysis of transgene expression.

Gene modification (transfection) of HEK293T and SH-SY5Y cell lines using the obtained pBud (Kan) -VEGF165-SDF-la genetic construct was performed using TurboFect transfection agent (Thermo Fisher Scientific Inc., USA) in accordance with the method recommended by the manufacturer.

To assess the expression of vegf and sdf-1 genes in transfected HEK293T and SH-SY5Y cells, immunofluorescence and western blot analyzes were performed.

Immunofluorescence analysis of SH-SY5Y cells transfected with pBud (Kan) -VEGF165-SDF-la plasmid revealed a positive reaction with rabbit polyclonal antibodies to vascular endothelial growth factor and stromal cell factor 1 (Fig. 1).

Protein lysates of HEK293T cells transfected with pBud (Kan) -VEGF165-SDF-1α plasmid were analyzed using Western blot analysis, which showed the presence of pronounced bands corresponding to the expected molecular weights of VEGF 165 and SDF-1α proteins of 42 kDa and 10 kDa, respectively ( Fig. 2).

After transfection of HEK293T cells with the pBud (Kan) -coVEGF165-coSDF1-α genetic construct using the TurboFect transfection agent (Thermo Fisher Scientific Inc.), the expression of the recombinant protein was analyzed by enzyme-linked immunosorbent assay (ELISA).

Collection of culture medium from wells with HEK-293A cells transfected with pBud (Kan) -coVEGF165-coSDF1-α was performed 1 day after transfection. The medium from the wells was transferred into 1.5 ml tubes and centrifuged for 1 minute at maximum speed to remove cell debris (debris). The supernatant was transferred into clean 1.5 ml tubes, diluted 10 times and used for enzyme-linked immunosorbent assay for determining the concentration of VEGF. The concentration of VEGF in the culture medium was determined using the VEGF-IFA-BEST A-8784 kit (Vector, Russia) according to the method recommended by the manufacturer. The determination method is based on a solid-phase "sandwich" variant of enzyme-linked immunosorbent assay using mono - and polyclonal antibodies to human VEGF. The range of measured concentrations is 0-2000 pg / ml, the sensitivity of the analysis is 10 pg / ml. The optical density in the wells was determined using an Infinite M200Pgo multifunctional microplate reader (Tesap) in a two-wave mode: main filter - 450 nm, reference filter - 655 nm. Based on direct proportionality between the optical density and the concentration of VEGF in standard samples, the concentration of VEGF in the test samples was calculated.

HEK293T cells transfected with pBud (Kan) -coVEGF165-coSDF1-α using TurboFect reagent showed a high level of VEGF expression 1 day after transfection (1542 pg / ml) compared to native (untransfected) cells (28 pg / ml).

Thus, the expression of VEGF 165 protein by the resulting pBud (Kan) -coVEGF 165-coSDF1-α genetic construct is shown.

To study the angiogenic efficacy of HEK293T cell secretion products transfected with pBud (Kan) -VEGF165-SDF-1α plasmid, the effect of the conditioned medium on the formation of capillary-like structures by HUVEC endothelial cells on Matrigel was evaluated (Matrigel ™ Matrix, BD Biosciences, San Diego, CA, USA ) [2].

It was shown that the conditioned medium of HEK293T cells genetically modified with the plasmid pBud (Kan) -VEGF 165-SDF-1α stimulates the formation of capillary-like structures in vitro by HUVEC cells (Fig. 3).

The resulting genetic engineering construct pBud (Kan) can be used to create genetic therapeutic drugs.

Recombinant plasmids created on the basis of the genetic engineering construct pBud (Kan) can be used as part of a pharmaceutical composition to obtain a finished dosage form.

A pharmaceutical composition for restoring blood supply to tissues and (or) organs contains the pBud (Kan) -coVEGF165-coSDF1-α gene engineering construct in an effective amount and pharmaceutically acceptable excipients.

In one embodiment, the pharmaceutical composition may include, inter alia, an additive for transfecting human cells (mononuclear cells of a blood or bone marrow of a person, mesenchymal stem cells, etc.) in the case of the implementation of the gene-cell approach and transplantation of gene-optimized cells.

A solution of pBud (Kan) -coVEGF165-coSDF1-α, suitable for the subsequent preparation of the pharmaceutical composition and formulation, can be obtained using standard DNA isolation and purification methods known to those skilled in the art.

The finished dosage form pBud (Kan) -coVEGF165-coSDF1-α should be suitable for conducting gene therapy and should not lead to a significant change in the properties of the main substance during prolonged storage. A possible finished dosage form of the plasmid can be selected from the group of frozen solution, liquid solution, lyophilisate, i.e. freeze-dried solution, but not limited to an amorphous film.

Preferred dosage forms are liquid solution or lyophilisate, as they can be stored at a positive temperature, i.e. in standard pharmaceutical refrigerators, and do not require significant time to prepare for injection.

A lyophilizate is the most preferred dosage form, since the absence of water potentially slows down the chemical reactions of DNA chain breakdown.

Obtaining a lyophilisate, i.e., an amorphous or microcrystalline porous mass, requires the presence of auxiliary substances in the lyophilized solution that perform the functions of a cryoprotectant, pH stabilizer, chelating agent, antioxidant, filler, etc. The smallest possible set of excipients may include at least one cryoprotectant having filler properties and a pH stabilizer. The excipient, which is a cryoprotectant and excipient, can be selected from the group comprising mono- and disaccharides, polyols and polymers, such as sucrose, lactose, trehalose, mannitol, sorbitol, glucose, raffinose, polyvinylpyrrolidone, or combinations thereof. The pH stabilizer may be selected from the group consisting of sodium citrate, sodium phosphate, Tris-HCl, Tris-acetate, glycine and other amino acids.

A method for producing a lyophilizate may include adding to the solution of purified plasmid DNA solutions of at least one cryoprotectant having filler properties and a pH stabilizer to obtain an isotonic solution with a concentration of purified plasmid DNA from 0.1 to 10 mg / ml and a pH of 7, 0 to 9.0 and subsequent lyophilization and storage at a temperature of + 2 ° C to + 8 ° C.

A method of using the pharmaceutical composition is to administer it to a person (or animals) in such a way and in such an amount that they will provide a therapeutic effect depending on the nosological form and medical indications.

The pharmaceutical composition can be administered locally - intramuscularly, subcutaneously, intradermally, systemically - intravenously, intraarterially, aerosolized, in the form of gene-cell transplantation or transfusion after in vitro treatment of various autologous cells, for example, hematopoietic and their more differentiated derivatives, mesenchymal, vascular-stromal fractions , mesangioblasts, myoblasts, myosatellite cells, etc.

Although these inventions are described in detail, for a person skilled in the art, it is obvious that various changes can be made and equivalent replacements made, and such changes and replacements are not beyond the scope of the present invention.

Currently, a number of similar constructions are used in the world, where cEGA of the VEGF165 gene is used as an insert, and various plasmids are used as a vector [3, 4].

Compared to the existing prototype [4], the pBud-VEGF165-FGF2 genetic construct, the pBud (Kan) -coVEGF165-coSDF1-α genetic engineering construct has the following advantages:

1. The genetic engineering construct of pBud (Kan) -coVEGF165-coSDF1-α contains vegf and sdf-1 genes optimized for the cDNA codon composition, which improves translation of the products of these genes and reduces the possibility of homologous recombination with corresponding sequences on human chromosomes.

2. The genetic engineering construct of pBud (Kan) -coVEGF165-coSDF1-α contains a clinically safe sequence of the antibiotic kanamycin resistance gene for plasmid selection in bacterial cells only.

3. Simultaneous independent expression of two factors VEGF165 and SDF1-α, controlled by two strong eukaryotic promoters CMV and EF1-α. Simultaneous expression of two genes in one cell allows a more uniform gradient of secreted recombinant proteins to be achieved. In addition, transfection of cells with pBud (Kan) -coVEGF165-coSDF1-α plasmid leads to higher expression of chemokines / cytokines IL-8 and MCP-1 compared to plasmid, with one veg / 165 gene (see Fig. 5).

Stimulation of the production of two genes simultaneously by a transfected cell — vegf and sdf-1 — leads to even higher expression of these genes, both introduced by the plasmid and endogenous. 12 days after transfection of the MMSC culture with the plasmid pBud (Kan) -coVEGF165-coSDF1-α, the concentration (accumulation) of the VEGF protein in the culture medium was statistically significantly higher than in the case of transfection of a similar culture with a plasmid with only one VEGF gene: 3064.61 ± 22 , 62 pg / ml and 2259.59 ± 171.56 pg / ml. Detailed intracellular mechanisms providing such a mutual potentiating effect of vegf and sdf-1 gene expression require further research, but the existence of such a phenomenon is confirmed by our studies. Its biological meaning is the synergistic effects provided by the VEGF and SDF-1 proteins. So, when implementing the processes of reparative tissue regeneration, VEGF stimulates the proliferation and differentiation of resident endothelial progenitor cells, their formation of capillary-like structures. At the same time, SDF-1 provides the migration of additional cambial cells, including endothelial progenitor cells and HSCs, according to the gradient of its concentration to the target zone in which they are affected by the VEGF protein. Thus, both therapeutic proteins work in pairs, stimulating angiogenesis and reparative regeneration. It is such a synergistic effect that could serve as the reason why the expression of these genes is in direct proportion, due, apparently, regulation of transcription, which has not yet been established by common intracellular pathways.

4. The pBud (Kan) genetic engineering construct does not contain the V5-His and myc-His tag sequences.

Thus, the construction of pBud (Kan) -coVEGF165-coSDF1-α has important advantages in terms of safety and efficiency of its use. The structure of the pBud (Kan) -coVEGF165-coSD F1-α construct is fundamentally different from all existing analogues.

Literature

1. Favaloro, L., M. Diez, et al. (2012). "High-dose plasmid-mediated VEGF gene transfer is safe in patients with severe ischemic heart disease (Genesis-I). A phase I, open-label, two-year follow-up trial." Catheter Cardiovasc Interv.

2. Construction of recombinant adeno-associated virus vector co-expressing hVEGF165 and hBMP-7 genes. Huang X., Shi Z., Wang K. et al. Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2008; 22 (7): 807-13.

3. Induced pluripotent stem cells generated from human adipose-derived stem cells using a non-viral polycistronic plasmid in feeder-free conditions. Qu X, Liu T, Song K, et al. Plos one. 2012; 7 (10): e48161.

4. EP 2520302, 11/07/2012.

5. Benton, G., J. George, et al. (2009). "Advancing science and technology via 3D culture on basement membrane matrix." J Cell Physiol 221 (1): 18-25.

6. Cherviakov Iu, V., I.N. Staroverov, et al. (2012). "[Therapeutic angiogenesis in treatment of patients with chronic obliterating diseases of lower limb arteries]." Angiol Sosud Khir 18 (3): 19-27.

7. Salafutdinov II, Sh.A.K., Yalvach M.E., Kudryashova HB, Lagarkova MA, Shutova MV, Kiselev SL., Masgutov RF, Zhdanov RI, Kiyasov A.P., Islamov PP, Rizvanov A.A. (2010). "The effect of the simultaneous expression of various isoforms of VEGF vascular endothelial growth factor and FGF2 major fibroblast growth factor on the proliferation of human umbilical vein endothelial cells HUVEC." Cell transplantology and tissue engineering V (2): 62-67.

Claims (4)

1. The recombinant plasmid for the preparation of a pharmaceutical composition, represented by SEQ ID NO: 1, into which the codon-optimized cDNA represented by SEQ ID NO: 2, from the HindIII and XbaI restriction sites, the codon-optimized cDNA represented by SEQ ID NO: 3. 2. Codon-optimized cDNA represented by SEQ ID NO: 2 encoding stromal cell factor 1 alpha. 3. Codon-optimized cDNA represented by SEQ ID NO: 3 encoding vascular endothelial growth factor isoform 165. 4. A pharmaceutical composition for repairing blood vessels and improving blood supply to damaged tissues or organs, comprising the recombinant plasmid according to claim 1 in an effective amount and pharmaceutically acceptable excipients.

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