WO2021091434A9 - Structure de génie génétique pour stimuler l'angiogenèse - Google Patents

Structure de génie génétique pour stimuler l'angiogenèse Download PDF

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WO2021091434A9
WO2021091434A9 PCT/RU2021/050002 RU2021050002W WO2021091434A9 WO 2021091434 A9 WO2021091434 A9 WO 2021091434A9 RU 2021050002 W RU2021050002 W RU 2021050002W WO 2021091434 A9 WO2021091434 A9 WO 2021091434A9
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gene
hgf
vegf165
plasmid
promoter
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WO2021091434A8 (fr
WO2021091434A3 (fr
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Екатерина Александровна СЛОБОДКИНА
Максим Николаевич КАРАГЯУР
Вадим Юрьевич Балабаньян
Елена Викторовна ПАРФЕНОВА
Павел Игоревич МАКАРЕВИЧ
Жанна Алексеевна АКОПЯН
Всеволод Арсеньевич ТКАЧУК
Мария Александровна БОЛДЫРЕВА
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Общество С Ограниченной Ответственностью "Генная И Клеточная Терапия"
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/515—Angiogenesic factors; Angiogenin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/4753—Hepatocyte growth factor; Scatter factor; Tumor cytotoxic factor II
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells

Definitions

  • the invention relates to the field of pharmaceuticals and biotechnology, and can be used to enhance the growth of new vessels in ischemic tissues.
  • the invention is a plasmid bicistronic construct carrying the hHGF gene of the human hepatocyte growth factor and the hVEGF165 gene of the vascular endothelial growth factor.
  • the claimed bicistronic construction can be used as a drug for medical purposes to stimulate angiogenesis, growth and remodeling of blood vessels, as well as restore blood supply in ischemic tissues, for the treatment of a number of human diseases caused by impaired blood supply to tissues.
  • CHD Ischemic heart disease
  • CLI critical ischemia of the lower extremities
  • Neovasculgen which is a plasmid encoding a vascular endothelial growth factor (pCMV-VEGF165)
  • pCMV-VEGF165 vascular endothelial growth factor
  • the active component of the drug is a plasmid with the hepatocyte growth factor gene - HGF, which has angiogenic properties; the drug has been reassigned for the treatment of critical ischemia of the lower extremities (CLLI).
  • angiogenesis is a multi-stage process, and each stage is under the control of various cytokines and growth factors; therefore, the use of only one angiogenic factor (both in the form of a recombinant protein and in the form of a genetic construct) does not allow achieving significant results. It is known to use in therapeutic angiogenesis the complex administration of several plasmids encoding different angiogenic growth factors (PRGs) in combination with each other.
  • PRGs angiogenic growth factors
  • An agent for stimulating angiogenesis in ischemic tissue is known from the prior art, which contains a mixture in 0.9% NaCl solution of plasmid constructs pC4W-hVEGFopt carrying the optimized vascular endothelial growth factor hVEGFopt gene and pC4W-hHGFopt carrying optimized hHGFopt gene of hepatocyte growth factor (RF patent N ° 2449799, 05/10/2012).
  • Effective angiogenesis in ischemic tissue is achieved using this agent in a mass ratio of the plasmid constructs pC4W-hVEGFopt and pC4W-hHGFopt from 1: 3 to 3: 1. The best results were achieved with a mass ratio of 1: 1.
  • Plasmids carrying IRES allow simultaneous expression of several genes; however, the translation efficiency provided by IRES is usually significantly inferior to cap-dependent initiation, and the final concentration ratio of the synthesized proteins varies depending on the selected IRES.
  • a group of Polish scientists conducted a clinical study of a plasmid construct carrying the VEGFA and FGF2 genes, separated by the IRES sequence of the EMCV virus, for the treatment of coronary heart disease [Kukula K, Chojnowska L, D ⁇ browski M, Witkowski A, Chmielak Z, Skwarek M, K ⁇ dziela J, Teresmska A, Malecki M, Janik P, et al.
  • VVF-CAD refractory coronary artery disease
  • plasmid construct in which the genes of the growth factors HGF and VEGF165 are separated by the site of internal ribosome entry - IRES [Slobodkina E.A., Nimiritsky P.P., Dolinkin A.O., Makarevich P. I., Parfyonova E.V. Development of a gene therapy plasmid construct encoding hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF165), Genes and Cells - Proceedings of the III National Congress on Regenerative Medicine. Moscow, November 15-18, 2017, series 3, place of publication Human Stem Cell Institute, Moscow, Russia Moscow, volume 12, abstracts].
  • HGF hepatocyte growth factor
  • VEGF165 vascular endothelial growth factor
  • the general scheme of this plasmid is shown in Fig. 1.
  • the genetically engineered construct is an expression vector plasmid into which cDNA inserts encoding vascular endothelial growth factor and hepatocyte growth factor are cloned, and contains:
  • Terminator - bGH poly A bovine growth hormone gene polyadenylation site
  • this plasmid does not provide the synthesis of sufficient amounts of angiogenic growth factors either in vitro, for example, the concentrations of HGF and VEGF165 in vitro (in the culture medium of HEK293T cells after their transfection) are no more than 2.72 nM and 1, 6 nM for HGF and VEGF165, respectively. At the same time, the concentration ratio of HGF and VEGF165 is unpredictable and on average ranges from 1:12 to 1: 5, depending on the IRES used. Also, this plasmid is not provides the synthesis of sufficient amounts of angiogenic growth factors neither in vitro nor when administered to mice - no more than 0.72 ng / ml and up to complete absence. Consequently, this vector does not have the parameters necessary for its use as an expression construct for the simultaneous expression of genes for hepatocyte growth factor and vascular endothelial growth factor.
  • a technical problem is the synthesis of two genes in a predetermined ratio, which provides effective stimulation of angiogenesis.
  • the technical problem is solved by the claimed plasmid vector, which carries the genes of human angiogenic growth factors and provides the synthesis of these AFR in vivo in a close to equimolar ratio.
  • the technical result is the creation of a plasmid genetic construct encoding hepatocyte growth factor (hHGF) cDNA and vascular endothelial growth factor (hVEGF165) cDNA under independent promoters, which makes it possible to synthesize these AFR in vivo at a concentration ratio of HGF / VEGF165 from 0.75 : 1 to 1: 2 while ensuring a stable yield of proteins.
  • the resulting vector proved to be effective and can be used to create a gene therapy preparation.
  • VEGF165 and HGF have their own promoters, enhancers and transcription terminators, the expression of each of them occurs independently and does not affect each other.
  • a vector the scheme of which is shown in Fig. 2, namely, a genetically engineered construct for stimulating angiogenesis in ischemic tissues, including:
  • SEQ ID NO: 1 is used as the sequence encoding the hepatocyte growth factor gene
  • SEQ ID NO: 2 is used as the sequence encoding the vascular endothelial growth factor gene.
  • the genetically engineered structure includes the following sequentially located elements:
  • Terminator 1 - bGH poly ⁇ (polyadenylation site of the bovine growth hormone gene) - 3044-3268 bp.
  • Promoter 2 - pCAG (chicken b-actin gene promoter) - 4742-5019 bp
  • Terminator 2 - SV40 poly A (polyadenylation site of the SV40 virus) - 5908-6029 bp
  • origin of replication (ori) is 6235-6823 bp.
  • the technical problem is also solved by the use of the claimed genetic engineering construct as an active substance of a gene therapeutic agent for stimulating angiogenesis in ischemic tissues.
  • the technical problem is solved by means for stimulating angiogenesis in ischemic tissues, including the claimed plasmid structure and auxiliary substances for intramuscular administration.
  • ischemic tissues including the claimed plasmid structure and auxiliary substances for intramuscular administration.
  • sodium chloride or water for injection were used as auxiliary substances.
  • the technical problem is also solved by a method for stimulating angiogenesis in ischemic tissues, including intramuscular administration of a drug for stimulating angiogenesis in ischemic tissues, including the claimed plasmid construct, in a therapeutically effective amount.
  • FIG. 1 shows a "map" of a prototype plasmid genetic construct encoding the hHGF gene of human hepatocyte growth factor and the hVEGF165 gene of vascular endothelial growth factor separated by the IRES sequence.
  • FIG. 2 shows a "map" of the plasmid genetic construct encoding the hHGF gene of the human hepatocyte growth factor and the hVEGF165 gene of the vascular endothelial growth factor under two independent promoters.
  • FIG. 3. is a diagram showing the content of HGF and VEGF165 in the HEK293T culture medium after calcium phosphate transfection.
  • FIG. 4. are photographs showing the formation of capillary-like structures by HUVEC cells on Matrigel after their treatment with experimental and control media.
  • FIG. 5 shows graphs showing the total length of capillary-like structures formed by HUVEC cells on Matrigel 15 hours after treatment with experimental and control media.
  • FIG. 6 shows graphs showing the number of branch points of capillary-like structures formed by HUVEC cells on Matrigel 15 hours after treatment with experimental and control media.
  • FIG. 7 shows the assessment of ischemic perfusion of the extremities in mice in the study groups.
  • FIG. 8 shows an analysis of the density of blood vessels on sections of the skeletal muscles of the animals of the studied groups.
  • the inventive genetically engineered construct is an expression vector plasmid into which cDNA inserts are cloned encoding vascular endothelial growth factor and human hepatocyte growth factor, namely, circular DNA with a length of 6888 nucleotide pairs.
  • cDNAs encoding vascular endothelial growth factor and human hepatocyte growth factor under independent promoters makes it possible to synthesize these AFR in vivo in a ratio of HGF / VEGF165 concentrations from 0.75: 1 to 1: 2 while ensuring a stable yield of proteins.
  • the plasmid construct carries optimized human VEGF165 (vascular endothelial growth factor) and HGF (hepatocyte growth factor) genes, which can be replaced with other (eg native) sequences of these genes.
  • VEGF165 vascular endothelial growth factor
  • HGF hepatocyte growth factor
  • Terminator 1 - bGH poly ⁇ (bovine growth hormone gene polyadenylation site) - 225 bp.
  • Terminator 2 - SV40 poly A (polyadenylation site of the SV40 virus) - 122 bp.
  • the plasmid was synthesized using standard technology and equipment used to solve similar problems in genetic engineering (Watson J. D., Gilman M., Witkowski J., Zoller M. - Recombinant DNA, Scientific American Books, New York, 1992).
  • any commercial vectors can be used, which are characterized by high-copy replication in E. coli (more than 150 copies per cell), a relatively small size (3-6 thousand bp) and a high level expression of the cloned gene in mammalian cells.
  • both codon-optimized sequences of genes of these PRA with the sequences SEQ ID NO: 1 and SEQ ID NO: 2, and native genes can be used.
  • competent strains of Escherichia coli for example, E. coli DH-5a.
  • VEGF165 vascular endothelial growth factor
  • HGF hepatocyte growth factor
  • Terminator 1 - bGH poly A bovine growth hormone gene polyadenylation site
  • Terminator 2 - SV40 poly A (polyadenylation site of the SV40 virus)
  • a selectable marker for the selection of transformed bacteria selected from the group including, but not limited to, antibiotic resistant markers such as ampicillin, kanamycin, neomycin, and the like.
  • the pharmaceutical composition based on the invention can be administered parenterally, intramuscularly, or in any other way that allows the compound and / or composition to be delivered to cells / tissues.
  • the pharmaceutical composition can be administered to a subject by intramuscular injection.
  • pharmaceutically acceptable refers to a non-toxic material that does not interact with the active ingredient of the pharmaceutical composition.
  • “Pharmaceutically acceptable carrier” refers to a biocompatible solution that is sufficiently sterile, pH, isotonic, stability, and the like, and may include any solvents, diluents, including sterile saline, sodium chloride injection, Ringer's solution for injection, dextrose solution for injection, solution of dextrose and sodium chloride for injection, lactated Ringer's solution for injection and other aqueous buffer solutions, dispersion media, coatings, antibacterial and antifungal agents, isotonic substances and the like.
  • the pharmaceutically acceptable carrier may also contain stabilizers, preservatives, antioxidants, or other additives that are well known to those skilled in the art, or other excipient known in the art.
  • concentration of the plasmid vector for highly efficient expression of the human VEGF165 and HGF genes in the carrier should be sufficient to allow therapeutic angiogenesis.
  • the therapeutic dose may be from about 25 to 1000 ⁇ g per kg of patient weight.
  • the introduction is recommended by intramuscular injection in a place as close as possible to the ischemic area. The introduction is performed after standard skin treatment, in compliance with the rules of asepsis, fractionally through several injections so that the entire muscle mass of the affected segment is infiltrated with a solution. The introduction can be carried out both once (once) or several times, depending on the indications, which is assessed by the attending physician based on the specific parameters of the patient, such as age, body weight, gender, degree of tissue damage, etc.
  • SEQ ID NO: 1 and SEQ ID NO: 2 were used as genes encoding VEGF165 and HGF. To increase the level of production of HGF and VEGF165 in transfected cells, the natural sequence of the cDNA of these genes was optimized.
  • the used design of the plasmid vector made it possible to accommodate cDNA of two growth factors in it without a significant increase in the size of the plasmid, and, on the other hand, made it possible to provide a close to optimal ratio of the produced proteins, i.e. high production of recombinant human-BDNF and moderate production of human-uPA.
  • the pVax2 expression vector used was constructed from the commercial pVaxl vector (ThermoFisher) and was taken as the main framework vector for the construction of the claimed invention.
  • the modification consisted of inserting the SV40 enhancer between the CMV promoter and the CMV pVaxl enhancer and replacing the kanamycin resistance gene KanR.
  • pVax2 Ceil was restirred (in buffer W + BSA) and the phosphorylated duplex was inserted to obtain the pVax2-VEGFIns construct.
  • Plasmid DNA isolated from about 10 of these colonies was analyzed for the presence of HGF and VEGF165 cDNA by restriction digestion using various restriction enzymes.
  • One such plasmid was sequenced using an automated DNA sequencer (ABI) and was determined to have the correct integration and sequence of the HGF and VEGF165 cDNA in vector pVax2.
  • a genetically engineered construct which is an expression vector plasmid (circular DNA with a length of 6888 bp), into which cDNA inserts encoding vascular endothelial growth factor and human hepatocyte growth factor are cloned (carries optimized genes VEGF165 (vascular endothelial growth factor ) and HGF (human hepatocyte growth factor), contains the main structural elements: • pCMV-HGF 488 - 2970 bp.
  • Terminator 1 - bGH poly ⁇ (polyadenylation site of the bovine growth hormone gene) - 3044-3268 bp.
  • Terminator 2 - SV40 poly A (polyadenylation site of the SV40 virus) - 5908-6029 bp.
  • origin of replication (ori) is 6235-6823 bp.
  • Selective marker for the selection of transformed bacteria - kanamycin resistance gene - 3441-4253 bp. (can be replaced with another antibiotic resistance gene or selectable marker).
  • E. coli bacteria strain DH-5a was used for amplification of all obtained plasmid DNAs. After transformation, bacteria were cultured overnight in LB selective medium with kanamycin at a concentration of 100 ⁇ g / ml. After the end of incubation, bacteria were precipitated by centrifugation and used to isolate plasmid DNA on a column (Qiagen, USA).
  • E. coli DH-5a strain was used for transformation and production of plasmid DNA (pDNA).
  • the original E. coli DH-5a strain is a museum and is kept in the All-Russian Collection of Industrial Microorganisms.
  • the transformed strain of E. coli DH-5a-CMV-HGF_CAG-VEGF165 differs from the original strain of E. coli DH-5a in resistance to the antibiotic kanamycin, provided by the plasmid “CMV-HGF_CAG-VEGF165” introduced into the strain.
  • Producer strain DH-5a-CMV-HGF_CAG-VEGF165 for the production of plasmid DNA was obtained by transformation of the original competent E.
  • E. coli strain DH-5a corresponding using plasmid, followed by selection of recombinant clones on LB medium with kanamycin at 37 ° C.
  • the transformation of competent cells of E. coli strain DH5a was carried out as follows. A suspension of bacteria thawed on ice (culture volume 30 ⁇ L) was added to an Eppendorf containing 1 yg of pDNA, after which transformation was carried out by the “heat shock” method. For this, the test tube was placed in a thermostat heated to 42 ° C for 45 sec, after which it was transferred to ice for 5 min.
  • sterile LB medium 500 ⁇ L was added to the resulting culture and incubated on a thermal shaker for 60 min (500 rpm, 37 ° C). Thereafter, 150 ⁇ l of the resulting suspension was transferred in a laminar bacteriological box onto pre-prepared Petri dishes 100 mm in diameter filled with LB agar containing the selection agent kanamycin (100 ⁇ g / ml). The transformation was controlled by a sample without pDNA addition, in which the absence of bacterial growth in LB agar with kanamycin was considered adequate.
  • the suspension was spread over the surface of LB agar using a burnt wire loop, and the resulting culture was transferred to an incubator for 15-17 h at a temperature of 37 ° C.
  • one colony of bacteria was transferred using a sterile spatula into a liquid culture of a small volume (15-20 ml, the ratio of the volume of liquid to air is not less than 1: 4-1: 5) on an LB medium containing 100 ⁇ g / ml of kanamycin.
  • the flask with the inoculated medium was placed in a thermal shaker for 18 hours (250 rpm, 37 ° C).
  • the resulting culture was transferred into a liquid LB medium with 100 ⁇ g / ml kanamycin in a volume of 500 ml in a ratio of 1: 500, after which it was incubated under similar conditions for 15-18 h to obtain a large volume of bacterial cells containing pDNA.
  • the resulting bacterial culture in LB medium (volume from 500 ml to 3 L, depending on the amount of pDNA) was precipitated by centrifugation (12000 g, 15 min), after which the liquid phase was removed, and the precipitate was used to isolate pDNA. Lysis, purification from endotoxins, and plasmid isolation were performed using Qiagen kits (Midi or Maxi EndoFree kit) according to the manufacturer's protocol.
  • VEGF165 and HGF concentrations in the HEK293T cell culture medium 48 h after transfection were 51.50 and 15.48 nM, respectively.
  • the HGF / VEGF165 molar concentration ratio was 0.3. The data obtained are shown in FIG. 3 3.
  • the plate with the cells was placed in the IncuCyte® system of in vivo analysis of cells, the cells were photographed in phase contrast mode with 100x magnification every 2 hours.
  • the length of the tubules and the number of branch points were estimated using the ImageJ software package. The result is shown in FIG. 4-6.
  • the needle of the insulin syringe was inserted percutaneously parallel to the longitudinal axis of the muscle, after which the solution was slowly injected into the thickness of the muscle to avoid rupture of perimisia.
  • the low-voltage electroporation method described by Schertzer and Lynch was used, with a minimal modification: the introduction of hyaluronidase, described in the original method, was omitted.
  • plate electrodes were applied, lubricated with a driving gel.
  • electroporation was carried out with three pulses with a voltage of 80 V / cm distance between the electrodes, a frequency of 1 Hz and a duration of 20 ms. Then the polarity of the electrodes was changed and three more pulses were supplied with similar characteristics given. After the electroporation was performed, the skin surface was cleaned with 70% ethyl alcohol.
  • VEGF165 and HGF were taken to assess the content of human VEGF165 and HGF by ELISA.
  • concentrations of VEGF165 and HGF produced by the murine muscle explants after injection were 0.81 and 0.35 ng / ml, respectively, on day 3 and 0.82 and 0.49 ng / ml on day 7.
  • the HGF / VEGF165 molar concentration ratio was 0.24 on day 3 and 0.32 on day 7.
  • plasmids carrying various IRES variants for simultaneous expression of the genes of the growth factors HGF and VEGF165 characterized by the general structure scheme: HGF-IRES-VEGF165.
  • HGF-IRES-VEGF165 To assess the production of VEGF165 and HGF, HEK293T cells were transfected with calcium by the phosphate method, the concentrations of VEGF165 and HGF in the cell medium were assessed by ELISA (the results are shown in Table 7).
  • HGF-IRES_EMCV-VEGF 165 carries the IRES of encephalomyocarditis virus (EMCV) as an IRES sequence.
  • the molar concentrations of HGF and VEGF165 were 1.48 and 0.28 nM, respectively.
  • the HGF / VEGF165 ratio was 5.42.
  • Plasmid N ° 2 - construct HGF-IRES_Bip-VEGF165 differs from construct N ° 1 in that the IRES sequence of the eukaryotic protein Bip is used as an IRES. Similar analyzes have shown that the concentrations of HGF and VEGF165 in the culture medium of plasmid-transfected HEK293T cells are 2.72 and 0.27 nM, respectively. The HGF / VEGF165 ratio was 10.7.
  • Plasmid N ° 3 - Construct HGF-IRES_FGF1-VEGF165 differs from Construct N ° 1 in that it includes the eukaryotic protein IRES sequence FGF1.
  • the molar concentrations of HGF and VEGF165 were 0.08 and 1.59 nM, respectively.
  • the HGF / VEGF165 ratio was 11.38.
  • VEGF165 and HGF proteins were evaluated in an explant culture model of skeletal muscle isolated after electroporation injection of vectors at point 1 (100 ng / ml) in mice. Samples of the culture medium for assessing the content of VEGF165 and HGF by ELISA were taken on the 3rd and 7th days of cultivation of the explants.
  • plasmid No. 1 the concentrations of HGF and VEGF165 produced by murine muscle explants were not detected by ELISA.
  • concentration of HGF in the culture medium of muscle explants was 0.72 ng / ml and 0.48 ng / ml on days 3 and 7, respectively.
  • the VEGF165 protein concentration was below the sensitivity threshold of the used detection method.
  • vectors N ° N ° 1, 2, 3 did not give sufficient AFR production for ELISA. From the data obtained, it can be concluded that the synthesized amounts of the VEGF165 and HGF proteins are lower than the dissociation constants of these AFRs with their receptors and are insufficient for the manifestation of their biological action.
  • multicistronic vector the following genetic construct was created and tested:
  • CMV cytomegalovirus
  • CAG chicken beta-actin
  • the HGF / VEGF165 ratio was 1.38.
  • Construct No. 4 was tested in vivo in the mouse skeletal muscle explant culture described in step 2.
  • the production of VEGF165 and HGF proteins was assessed by ELISA.
  • the HGF concentrations in the culture medium of muscle explants were 1.14 ng / ml and 1.3 ng / ml on days 3 and 7, respectively.
  • VEGF165 concentrations were 0.5 ng / ml and 0.2 ng / ml on days 3 and 7, respectively.
  • the HGF / VEGF165 ratio was 2.28 on day 3 and 6.67 on day 7 of explant culture.
  • the claimed genetic construct “pHGF / VEGF” was constructed.
  • the vector is a construct similar to plasmid No. 4, characterized in that the HGF gene sequence is replaced with an optimized one.
  • concentrations of the HGF and VEGF165 proteins in the culture medium of the plasmid-transfected HEK293T cells were 15.48 and 51.5 nM, respectively.
  • the HGF / VEGF165 ratio was 0.3.
  • mice To model ischemia of the hind limb, male C57 / B6 mice at the age of 8-10 weeks were used. Before the operation, the mice were anesthetized with an intraperitoneal injection of 300 ⁇ l of a 2.5% solution of avertin.
  • a model of mouse hind limb ischemia developed in earlier works of our team (Makarevich R., Tsokolaeva Z., Shevelev A., Rybalkin F, Shevchenko E., Beloglazova L, Vlasik T., Tkachuk V., Parfyonova Y. (2012) Combined transfer of human VEGF165 and HGF genes renders potent angiogenic effect in ischemic skeletal muscle.PLoS One).
  • the skin was incised along the midline of the thigh of the left hind limb, a. femoralis and its large branches were ligated distal to the inguinal ligament and proximal to its popliteal bifurcation.
  • the vessel was excised between the upper and lower ligatures. After stopping the bleeding, the skin was sutured with an atraumatic needle (5-0 silk) with a continuous suture. All surgical procedures were performed under aseptic conditions using a binocular microscope. After the operation was completed, to compensate for blood loss, all animals were injected subcutaneously with 1.5 ml of warm sterile saline solution and placed in a separate cage until complete recovery from anesthesia. Animals were randomized to receive experimental or control solutions into the following treatment groups:
  • the plasmids were diluted with sterile saline.
  • the solutions were injected in three equal injections (50 ⁇ l each) into the anterior tibia muscle, femoral biceps muscle, and femoral quadriceps muscle.
  • tibialis anterior was fixed on glass slides with acetone cooled to -200C for 20 minutes, dried and washed with PBS solution for 5 minutes. Slides were blocked with 10% normal donkey serum (30 min), washed, and incubated overnight with primary antibodies (rat anti-mouse CD31 antibodies, # 553370 BD Biosciences Pharmingen, San Diego, California, USA; rabbit antibodies against a- SMA, # 56945 Abeam., Cambridge, MA, USA) diluted with blocking solution (1% BSA in PBS).
  • primary antibodies rat anti-mouse CD31 antibodies, # 553370 BD Biosciences Pharmingen, San Diego, California, USA
  • the sections were then stained with a secondary antibody conjugated to Alexa Fluor® 488 (# A21206, Thermo Scientific, Waltham, MA, USA) or a secondary antibody conjugated to Alexa Fluor® 594 (# A21209, Thermo Scientific, Waltham, MA, USA) ( 1: 800) for 1 hour; all slides were contrasted with DAPI (40,6-diamidino-2-phenylindole) dye (Sigma-Aldrich, Milwaukee, WI, USA). Micrographs of the sections were taken at 200x magnification in 5 random fields of view using a Zeiss Axio Observer A1 (Zeiss, Oberkochen, Germany).
  • the resulting images were analyzed using the ImageJ software (ImageJ, NIH, USA).
  • the number of CD31-positive and a-SMA-positive structures was counted in 2-3 fields of view from 5-6 slices for each sample, after which the average number of structures per field of view for each animal was calculated. and for each study group.
  • significantly more CD31 + capillaries with a lumen were found in the pHGF / VEGF group than in the negative control group with saline: 30.37 ⁇ 4.26 versus 16.54 ⁇ 2.30, respectively (Fig. 8, A ).
  • a moderate increase in CD31 + vessels without lumen was found in the pHGF / VEGF group, but this increase was not statistically significant.
  • the only vector that allows one to achieve optimal concentrations and ratios of PRA is the claimed construct with two separate promoters for each of the genes: CMV for the HGF gene and CAG for the VEGF165 gene.
  • CMV for the HGF gene
  • CAG for the VEGF165 gene.
  • the use of two separate cassettes with their own enhancers, promoters, and transcription terminators for each of the genes provides independent expression of each of them.
  • the results of experiments with the vector selected as a prototype - plasmid No. 4, as well as with the inventive construct showed the activity of both promoters both in vitro in human cells and in vivo in an explant muscle culture obtained after plasmid injection mice.
  • inventive vector ensures the production of HGF and VEGF165 proteins in a close to equimolar ratio with a slight predominance of “angiogenesis activator” - vascular endothelial growth factor.
  • All variants of vectors based on excellent implementation methods do not allow achieving the technical result and cannot be used for the simultaneous in vivo expression of two angiogenic growth factors (PRGs) from one genetic construct in sufficient amounts and in an optimal ratio.
  • PRGs angiogenic growth factors

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Abstract

L'invention se rapporte au domaine des biotechnologies, concerne plus précisément des structures de génie génétique pour stimuler l'angiogenèse, et peut être utilisée en médecine afin de traiter des maladies liées à un trouble de l'apport en sang des tissus. L'invention concerne une structure bicistronique plasmide porteuse du gène hHGF de facteur de croissance d'hépatites humain et du gène hVEGF165 de facteur de croissance endothéliale des vaisseaux avec deux promoteurs distincts pour chacun des gènes: promoteur de citomégalovirus pCMV pour le gène HGF et promoteur de gène de β-actine du poussin pCAG pour le gène VEGF165. Cette structure bicistronique peut être utilisée en qualité d'agent médicamenteux afin d'assurer la stimulation de l'angiogenèse, la croissance et le remodelage de vaisseaux, ainsi que la restauration de l'alimentation en sang dans des tissus ischémiques. Cette invention permet d'atteindre des concentrations optimales et une corrélation des facteurs angiogènes de croissance (FAC).
PCT/RU2021/050002 2019-11-06 2021-01-05 Structure de génie génétique pour stimuler l'angiogenèse WO2021091434A2 (fr)

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RU2019135603A RU2737487C1 (ru) 2019-11-06 2019-11-06 Генно-инженерная конструкция для стимуляции ангиогенеза
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AU2001291019A1 (en) * 2000-09-15 2002-03-26 Genvec, Inc. Method of modulating neovascularization
CN1204247C (zh) * 2001-06-15 2005-06-01 中国人民解放军军事医学科学院百环生物医学研究中心 一种重组腺病毒及其在心肌缺血治疗中的应用
RU2449799C2 (ru) * 2009-10-30 2012-05-10 Федеральное Государственное Учреждение "Российский кардиологический научно-производственный комплекс Федерального агентства по высокотехнологичной медицинской помощи" Средство для осуществления терапевтического ангиогенеза и способ его осуществления
CN102242148A (zh) * 2010-10-09 2011-11-16 苏州大学 重组载体以及转基因骨髓基质细胞修饰的丝素膜及其应用

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