RU2263146C2 - Method for preparing bacteriophages binding specifically with target-cells and designated for therapeutic aims - Google Patents

Abstract

FIELD: medicine, genetic engineering.

SUBSTANCE: invention proposes a method that involves construction of bacteriophage library of random peptides based on oligonuleotide fragments encoding their, selection of bacteriophages binding with target-cells but not binding with cells of other types that can be involves in pathological process or able to show effect on its diagnosis and therapy, and confirmation of specificity of selected bacteriophages by using combination of different tests. Oligonucleotide fragments encoding random peptides are prepared by reaction of reverse transcription by using random primers and total RNAs isolated from indicated target-cells and cells of other types. Applying this invention provides preparing bacteriophages binding with target-cells with high degree of selectivity. Invention can be used in diagnosis, therapy and pharmaceutical industry.

EFFECT: improved preparing method.

3 cl, 2 dwg, 8 ex

Description

The invention relates to genetic engineering, in particular to methods for producing bacteriophages that are able to specifically bind to target cells and can be used in diagnostics, therapy, and pharmaceutical applications.

Currently, one of the main problems is to increase the level of cardiovascular, cancer and immune diseases. In most cases, the primary sources of such disorders are transient uncontrolled proliferation of tissue-cell structures, which complicates and complicates their diagnosis and therapy. Therefore, the primary condition is the timely identification of the pathological condition, the development and delivery of drugs that block cellular activity in the foci of diseases.

Known therapeutic inhibitors for vascular smooth muscle cells, which are used to suppress cell activity and / or cell destruction by targeted delivery of therapeutic agents to foci of active proliferation (see US Pat. No. 6515009 of 02.02.2003, IPC A 61 K 31/40 ) For the inhibition of vascular smooth muscle cells, according to this patent, therapeutic conjugates are used that contain a drug associated with a peptide or protein that specifically binds to the cell membrane of vascular smooth muscle cells (e.g., monoclonal and polyclonal antibodies, growth factors, polypeptide hormones, cytokines and similar) or with an element of the intracellular or extracellular matrix (for example, macromolecules that recognize integrin and fibronectin receptors, and the epitopes of collagen and extracellular glycoproteins) of the same cells. One of these peptides, for example, contains an amino acid sequence that specifically recognizes chondroitin - proteoglycan sulfate, with an estimated molecular weight of about 250 kilodaltons, expressed on the membranes of vascular smooth muscle cells. The same amino acid sequence is found in Fab and Fv fragments or CDRs (regions for complementarity) of the monoclonal antibody NR-AN-01 and / or its functional equivalents.

The disadvantages of this invention is the use of peptides that recognize proteins with pronounced expression in a number of other functionally important cells and tissues, this leads to the fact that this therapeutic conjugate can bind to other types of cells of non-smooth muscle origin, such as neuroglia, bone cells tissues and others, which leads to undesirable accumulation of the drug in these cells and a side cytotoxic effect (cell death). In addition, when antibodies are used as the leading constituents of the conjugates, their size, due to the dense extracellular matrix, can become an obstacle to the penetration of the conjugate into the proliferation site, and the cross-reactivity of antibodies to other molecular structures reduces their specificity.

There is also known a method for targeted delivery of peptides to restenous vascular smooth muscle cells in vitro and in vivo with the aim of delivering drugs to restenous plaque via peptides built into phage particles that bind to proliferating vascular smooth muscle cells from a library of synthetic 15 membered peptides constructed by random way (see Ingrid N. Michon et al., “Targeting of peptides to restenotic vascular smooth muscle cells using phage display in vitro and in vivo” in Biochimica et Biophysica Acta, issue No. 1591, 2002, p. 87-97). The selected peptides by repeated repetition of one-step screening subsequently pass the confirmation of specificity exclusively by enzyme-linked immunosorbent assay and visualized using immunofluorescence, in which two peptides with a conservative amino acid sequence, such as 5G6 (CNIWGVVLSWIGVFPEC) and 5Е5 (CESLWGDCL) are detected.

The disadvantages of this method are the use of a single source library based on synthetic randomly oriented peptides that have no analogues in nature, which determines their initially low specificity and selectivity, as well as high immunogenicity. In addition, repeated repetition of one-stage screening leads to weakening of specific interactions and the growth of non-specific ones.

The basis of the claimed invention is tasked with creating a method for producing bacteriophages that specifically bind to target cells with a high degree of selectivity, which are intended for therapeutic purposes.

The problem is achieved by implementing a new method for producing bacteriophages that specifically bind to target cells, which involves the construction of a phage library of random peptides based on the oligonucleotide fragments encoding them, screening it to obtain bacteriophages that bind to target cells, and confirm the specificity of the selected bacteriophages, however, according to the invention,

- oligonucleotide fragments encoding random peptides are obtained by the reverse transcription reaction using random primers and total RNA of target cells and other types of cells that may be involved in the pathological process or capable of influencing its diagnosis and therapy;

- these oligonucleotide fragments are inserted into the bacteriophage vectors in the correct orientation and used to create phage libraries of random peptides for all types of cells used to isolate total RNA;

- screening of the obtained phage libraries of random peptides is carried out in two stages, in the first of which bacteriophages capable of binding to target cells are selected, and in the second, non-binding to cells of other types used are selected from the bacteriophages selected in the first stage;

- to confirm the specificity of bacteriophages to target cells, a combination of various tests is used.

Moreover, vascular smooth muscle cells (HCMCs) are chosen as target cells, and the criterion for the selection (screening) of specific bacteriophages is their ability to bind to such cells and not bind to other cell types. In addition, vectors based on T7 bacteriophage DNA are used as bacteriophage vectors.

When implementing this method, bacteriophages with a high degree of selectivity are obtained, which can be used to deliver drugs to their place of direct action for the prevention and / or therapy associated with impaired functioning of vascular smooth muscle cells, for the direct use of the obtained peptides as a dosage form in therapeutic applications . In addition, the use of such bacteriophages or their fragments, or a modified vector will allow to diagnose a malfunction of vascular smooth muscle cells and predict their treatment.

The claimed method allows to obtain highly selective bacteriophages to a specific type of target cells, namely to vascular smooth muscle cells.

The method of obtaining bacteriophages is illustrated by diagrams and illustrations, where

figure 1 shows the synthesis of cDNA and construction of libraries;

figure 2 shows a diagram of single and multi-step screening.

A method of producing bacteriophages is carried out in this way.

First, the selection and separation of cell populations into types is carried out: target cells and cells that may be involved in the pathological process or capable of influencing its course, diagnosis and therapy. Then construct a series of libraries of random peptides using total cellular RNA in a viral type vector based on bacteriophage DNA.

First, total RNA is isolated by well-known methods with adaptive modifications, followed by, if necessary, purification of it and isolation of a fraction of messenger RNA. The quality of such RNA is further analyzed, RNA is used in reverse transcription reactions directed by primers, both of random origin and complementary to the poly A + region of messenger RNAs.

At this stage, the primary separation of total cellular RNA is carried out by controlled synthesis of cDNA fragments, limited in size and heterogeneity, which is achieved by using different concentrations of ions in the reaction mixture, a combination of primers, the amount of starting material and temperature conditions.

Secondary separation of cellular RNA is carried out at the stage of fractionation and purification of the obtained cDNA molecules, for which gel filtration chromatography is used so that after elution the pool of cDNA molecules consists of 2 parts: a fraction with the number of nucleotide units less than 500 and a fraction consisting of molecules larger than 500 base pairs.

This separation allows, in the first case, the creation of libraries aimed at determining the exact places of interaction of the peptide molecule, as part of the phage particle, with the molecules on the target cells, and in the second case, the nucleic acid fragment responsible for the synthesis of this peptide or protein fragment can be identified.

Further, at the stage of embedding cDNA fragments into the bacteriophage vector in the correct orientation, the cDNA molecules obtained by division and synthesis are ligated with oligonucleotide linkers synthetically synthesized and containing recognition sites for restriction endonucleases (restrictase enzymes) used to process and prepare the vector for cloning. For this, the ends of the cDNA are first processed using polymerase and only then such fragments are ligated with linkers and incubated with restrictase enzymes. Using these linkers, it becomes possible to obtain cDNA fragments that contain recognition sites for the used restriction enzymes at different ends in such an arrangement that they can be freely cloned into a vector in a specific orientation (semantic cloning).

At the same time, the bacteriophage vector is treated with restriction endonucleases in order to obtain fragments suitable for ligation with the synthesized cDNA fragments. The recombinant molecules resulting from ligation of cDNA fragments and vector molecules are packaged in vitro and the expression of this fragment is initiated through a single replication cycle. Then determine the number of recombinants in the library and the absolute number of particles.

After that, selection (screening) of such libraries is carried out using combinations of target cells and cells that may be involved in the pathological process or capable of influencing its course, diagnosis, and therapy. In this case, the screening is carried out by repeated repetition of incubation rounds with cells, washing, solutions of different ionic strength and solutions of different ability to disrupt protein - protein interactions, phage particles that did not bind to cells, followed by elution and amplification of the desired phage particles. Moreover, in the case of direct screening, it is certain that the calculated amount of phage particles in the buffer is first incubated with the material used for cell cultivation, and then the residual fraction (supernatant) containing free phages is transferred to the target cells. In the case of multi-step screening, namely the use of combinations of several types of cells that may be involved in the pathological process or may affect its course, diagnosis and therapy, the supernatant after incubation with the material is sent to these cells, after which the supernatant is sent either to the cells- target, or on the next type of cells that may be involved in the pathological process or capable of influencing its course, diagnosis and therapy, followed by incubation on target cells . Similarly, when using target cells and cells that can be involved in the pathological process or are able to influence its course, diagnosis and therapy, in various structural and functional states.

Thus, the result of multistep screening is a group of peptides composed of phage particles, which has a pronounced specificity for target cells.

To obtain the final bacteriophages from the above groups, individual phage particles are isolated and tested.

In one case, the test phage is produced (increased) in the required amount, purified and tested as a primary antibody in a direct enzyme-linked immunosorbent assay (ELISA), where the cultured cells of the selected functional state act as an antigen.

In the study of a large number of phage particles, microselection is performed. To do this, the initial group is scattered to obtain individual plaques, which are then eluted with the buffer for incubation in ELISA and used directly as a source of primary antibodies in ELISA, without preliminary growth and purification.

In another case, an individual clone is amplified, purified and incubated with target cells and cells that can be involved in the pathological process or capable of influencing its course, diagnosis, and therapy, at the same time as the first case. Then the cells are washed, bound particles elute and their titer is determined, then they are compared with the same for another type of cells.

In the third case, polymerase chain reaction (PCR) is used to analyze the specificity of peptides, while several phages are purified and mixed in equal amounts, then incubated with target cells and cells that may be involved in the pathological process or can affect its course, diagnostics and therapy, elute, scatter to individual plaques that are selected in pairs, DNA is isolated and used as a matrix for PCR. This determines the quantitative distribution and degree of specificity of phages.

Also determine the stability and ability to internalize, which is checked by incubating the phage with target cells at various temperature and time modes, followed by elution of the remaining phages.

Examples of specific performance of the claimed method.

Example 1. The choice of cell populations.

To obtain highly specific bacteriophages to vascular smooth muscle cells (VHCM), which are used as target cells, and the cells interacting with them, both as part of the blood vessel wall (endothelial) and transported by the blood stream (fibroblasts), are used as cells that may be involved in the pathological process or capable of influencing its course, diagnosis and therapy. Cervical canal carcinoma cells - HeLa and liver carcinoma cells - Hep-G2 are also used as cells that can be involved in the pathological process or can influence its course, diagnosis and therapy.

The culture of HCMC is initiated from the main blood vessels of patients with pathologies of the cardiovascular system. A number of cell lines are available as commercial preparations. For example, coronary artery smooth muscle cells (Cell Systems GmbH, Lot # 17151, Catalog # CC-2583) were used in parallel with cell cultures obtained by treating the arteries and veins of patients. Preparations of the initial culture of SGMK are primary in their origin and have a limited number of duplications, which limits the number of subsequent transfers. Therefore, in all cases, cultures are initiated for subsequent research using cell samples having no more than 6 divisions. For cultivation, plastic bottles are used, made exclusively for working with tissue cultures, with a total surface area for growth of 25 to 182 square centimeters (for example, bottles of types T25, T75, T182).

Vascular smooth muscle cells are cultured using a growth medium that consists of 1/3 of each of the following components - Waymouth (GIBCO BRL Cat. # 31220-023), F12 (GIBCO BRL Cat. # 31220-023), SMCBM (Promocell Cat. # C-22260). This medium is supplemented with a pre-inactivated incubation for 30 minutes at a temperature of 56 ° C in a water bath, cow fetal serum (GIBCO BRL Cat. # CC-4102) to a final concentration of 15%, as well as a solution of penicillin and streptomycin (GIBCO BRL Cat. # 15140 -114) to a final concentration of 1.5%. A medium containing cow fetal serum is called enriched. Vascular smooth muscle cells blocked at the growth stage are cultured using a depleted growth medium, i.e. without the addition of serum to the fetal cow, but with the addition of a solution of penicillin / streptomycin in the above concentration. To block vascular smooth muscle cells at the growth stage, they are first cultivated until the monolayer reaches 90% of the entire growth area using an enriched nutrient medium, and only then the enriched nutrient medium is replaced with a depleted one and incubation is continued for 72 hours, replacing the old one culture medium for a new one every 24 hours.

Endothelial cells are cultured using nutrient medium — EGM-2 (Clonetics Company, Cat. # CC-3202), supplemented with the complementation package EGM-2 MV (Clonetics Company, Cat # CC-4147) that contains growth factors, hormones and inactivated serum of a cow fetus, the final concentration of which in the medium after complementation is 10%. Endothelial cells are initiated from the drug (Promocell Cat. # CC-2519). Block endothelial cells at the growth stage in the same way as in the case of vascular smooth muscle cells.

Fibroblasts are cultured using a nutrient medium based on standard DMEM medium (GIBCO BRL) supplemented with inactivated bovine fetal serum to a final concentration of 10% and penicillin / streptomycin solution to a final concentration of 1.5%. Initiate a fibroblast cell culture from a preparation of patients with pathologies of the cardiovascular system and block in the same way as described for vascular smooth muscle cells.

HeLa 3T3 cells and Hep-G2 cells are immortalized lines; therefore, almost any available cell sample is used to initiate culture, however, they try to use cells that have passed no more than 100 divisions. For the cultivation of these cells using a nutrient medium based on any of the MEM compositions (GIBCO BRL), supplemented with fetal serum of a cow at a final concentration of 15-20% (which is determined by the desired growth rates) and a solution of penicillin / streptomycin at a final concentration of 1.5 % To obtain reliable screening results, these cell types are also cultured using a depleted nutrient medium, simulating growth blocking, similar to that observed for primary cell types (SGMK, endothelial and fibroblasts).

Example 2. Isolation of RNA and reverse transcription reaction (see figure 1).

Based on the initial selection of cell populations, RNA is isolated from both target cells and cells that may be involved in the pathological process or capable of influencing its course, diagnosis, and therapy.

Cells of all types are grown within 5-7 days, using T75 bottles and 18 ml of culture medium, which are replaced every day. Vascular smooth muscle cells are cultured in 40 vials, at the rate of 3.5 · 10 ⁵ cells per vial. Endothelial cells are cultured in 20 vials, at the rate of 3 · 10 ⁵ cells per vial. HeLa and Hep-G2 cells are cultured in 20 vials, at a rate of 1.6 · 10 ⁵ cells per vial. Cells are washed extensively 2 times using 12 ml of HBSS buffer (GIBCO BRL) each time, then treated for 3 minutes using 6 ml of a solution containing 0.05% trypsin and 0.53 mm EDTA (ethylenediaminetetraacetate) and neutralized by the addition of 9 ml HBSS buffer. Cells are harvested by centrifugation for 10 minutes at 3000 * g and washed 3 times using 20 ml of HBSS buffer. The number of cells is determined using a cell counting chamber; for this, 50 μl of a cell suspension are mixed with an equal volume of a 15% trypan blue solution, which is a vital dye. The number of living cells is fixed and the total number of cells in suspension is calculated.

In the case of coarse extraction methods with guanidine isothiocyanate, both followed by precipitation with lithium salts and without precipitation, the cells are concentrated by centrifugation, the supernatant is removed, and the pellet is resuspended intensively in lysis buffer (5.25 M guanidine isothiocyanate; 50 mM Tris-Cl, pH 6, 4; 20 mM EDTA; 1% Triton X-100; 0.1 M 2-mercaptoethanol) at a rate of 1 ml per 10 ⁷ cells. To this solution add 0.1 volume of 2 M solution of sodium acetate, 1 volume of solution of aqueous phenol, 0.3 volume of a mixture of chloroform - isoamyl alcohol (in a ratio of 24: 1) and mix intensively for 30-60 seconds. Incubate the resulting mixture for 15 minutes at a temperature of 0 ° C, and then centrifuged for 30 minutes at 8000 * g at a temperature of 4 ° C in order to separate the aqueous and organic phases. After precipitation, the aqueous phase is transferred to a new test tube and 0.6 volumes of isopropyl alcohol pre-cooled to -20 ° C are added to it, mixed and placed for at least 60 minutes in a cooler with a temperature of -70 ° C. After incubation, the solution is centrifuged for 30 minutes at 12000 * g at 4 ° C, the supernatant is thoroughly removed, and the precipitate is washed several times by centrifugation for 5 minutes in the presence of 1 ml of 70% ethanol. The resulting precipitate was dissolved in 0.4 ml of water treated with diethyl pyrocarbonate (DEPC).

In the case of applying soft extraction methods using proteinase K, the cells are concentrated by centrifugation, the supernatant is removed, and the pellet is resuspended intensively in pre-chilled lysis buffer (0.15 M sodium chloride; 10 mm Tris-Cl, pH 8.5; 0.05 % NP-40; 0.01% 3-carboxyauric acid; 0.1 M 2-mercaptoethanol) at the rate of 1 ml per 5 · 10 ⁷ cells. Then the mixture is centrifuged for 2 minutes at 12000 * g and a temperature of 4 ° C, the supernatant is transferred to a new tube and an equal volume of proteinase K buffer is added (0.2 M Tris-Acetate; 0.3 M sodium acetate; 0.05 M EDTA, pH 7.5; 2% (w / v) sodium dodecyl sulfate) containing 400 mg / ml proteinase K and incubated for 60 minutes at 37 ° C. Then add 1 volume of a mixture of phenol-chloroform-isoamyl alcohol (in a ratio of 25: 24: 1) and mix vigorously for 5 minutes at room temperature. After which the mixture is centrifuged for 2 minutes at 12000 * g at room temperature, the resulting aqueous phase is transferred to a new tube and the extraction is repeated with a mixture of phenol-chloroform-isoamyl alcohol (in a ratio of 25: 24: 1) several times. 2.5 volumes of absolute ethanol are added to the aqueous phase obtained as a result of extraction, RNA is intensively mixed and precipitated by placing the sample for 30 minutes in a cooler with a temperature of -70 ° C. Next, the solution containing RNA is centrifuged for 10 minutes at 12000 * g at 4 ° C, and the resulting precipitate is washed 3 times by centrifugation for 5 minutes at 12000 * g in the presence of 700 μl of a 70% solution of ethyl alcohol. The precipitate was dried for 2.5 minutes under vacuum and dissolved in 0.4 ml of water treated with DEPC.

The solution of total cellular RNA obtained as a result of primary extraction is subjected to treatment with a deoxyribonuclease of the first type in order to remove DNA fragments that can get into the RNA solution during its extraction and can impede further fractionation of such RNA and its use in reverse transcription reactions. For this, 12 μl (480 units) of ribonuclease inhibitor (Invitrogen, Cat # 10777-019), 50 μl of a 10-fold standard deoxyribonuclease buffer and 50 μl (500 units) are added to 0.4 ml of a solution containing 4 mg / ml RNA. ) deoxyribonuclease of the first type (GIBCO BRL). Then the resulting mixture was incubated for 2 hours at a temperature of 37 ° C, and the reaction was stopped by the simultaneous addition of 0.1 volume of 3 M solution of sodium acetate (pH 5.2), 1.1 volume of a solution of aqueous phenol, 0.3 volume of a mixture of chloroform isoamyl alcohol (in a ratio of 24: 1). The solution was stirred vigorously for 1 minute, incubated for 15 minutes at 0 ° C and centrifuged for 10 minutes at 12,000 * g at 4 ° C. The aqueous phase is transferred to a new tube and RNA is precipitated with absolute ethanol, as described above.

To isolate the fraction of messenger RNA (mRNA), 1-2 mg of total RNA is dissolved in 5 ml of application buffer (attached to the column) and applied to a column containing oligo- (dT) -cellulose (Sigma, Cat # 03131), previously washed 3 times 1 ml of neutralization buffer (attached to the column). After extraction, the column is washed 5 times using 1 ml of neutralization buffer, and the bound mRNA is eluted with 1.5 ml of elution buffer. Subsequently, the mRNA contained in the resulting solution was precipitated by the addition of 150 μl of a solution of 3 M sodium acetate (pH 5.2) and 3.75 ml of absolute ethanol.

Example 3. Synthesis of cDNA and fragmentation of total RNA.

In order to obtain cDNA molecules for cloning, total RNA and / or mRNA is used in reverse transcription reactions directed by an RNA-dependent DNA polymerase.

To do this, mix 0.001-5,000 mg of total RNA and 1 μl of primer (500 μg of oligo- (dT) or 0.050-0.250 μg of random binding primer, 5'-TTNNNNNN-3 '), to which 1 μl of 10 mM deoxyribonucleotide solution is added (10 mm each in a neutral solution) and the volume of the mixture was adjusted to 12 μl using twice distilled water treated with diethyl pyrocarbonate. The resulting mixture was incubated for 5 minutes at 65 ° C, quickly cooled and 4 μl of 5-fold cDNA primary chain synthesis buffer (250 mM Tris-Cl, pH 8.3; 375 mM potassium chloride; 15 mM magnesium chloride) was added, 2 μl of 0.1 M solution of dithiotriethol (DTT), 1 μl (40 units) of ribonuclease inhibitor. The contents of the tube are vigorously mixed and incubated for 2 minutes at 42 ° C, after which 1 μl (200 units) of Superscript II RNA-dependent DNA polymerase (GIBCO BRL, Cat. # 18064-014) is added, vigorously stirred and incubated for 60 minutes at a temperature of 42 ° C. The reverse transcription reaction is stopped by inactivation of the enzyme, for which the mixture is incubated for 15 minutes at a temperature of 70 ° C. In order to remove RNA fragments complementary to newly synthesized cDNA molecules and hindering subsequent amplification of cDNA by PCR, 1 μl (2 units) of ribonuclease H was added to the solution and incubated for 20 minutes at 37 ° C.

At this stage, the primary separation of total RNA is carried out by controlled synthesis of cDNA fragments, limited in size and heterogeneity, which is achieved by using different concentrations of ions in the reaction mixture, a combination of primers, the amount of starting material and temperature conditions.

The synthesis of the 2nd cDNA chain is carried out using polymerase chain reaction (PCR), for which no more than 10% of the final solution is used after treatment with ribonuclease N. The mixture is collected by the sequential addition of 5 μl of 10-fold PCR buffer (200 mM Tris-Cl, pH 8.4; 500 mM potassium chloride), 1.5 μl of 50 mm solution of magnesium chloride, 1 μl of 10 mm solution of deoxyribonucleotides, 0.5 μl (2.5 units) of Taq DNA polymerase, 2 μl cDNA solution (the final product of the synthesis reaction of the primary cDNA chain), 38.1 μl of water treated with DEPC. The solution is intensively mixed and incubated for 2 minutes at a temperature of 94 ° C. Then spend 15-40 standard cycles of PCR amplification.

In order to obtain fragments suitable for cloning, the ends of the cDNA molecules are processed and ligated with synthetic oligonucleotide linkers, which contain the recognition site for the restriction endonuclease - Eco R1 and partially the recognition site for the restriction endonuclease - Hind III. This makes it possible to obtain, after treatment of such linkers with appropriate restriction endonucleases, cDNA molecules capable of efficiently ligating from the 5 ′ end only with Eco R1 treated DNA and from the 3 ′ end only with Hind III treated DNA. For this, 3 μl of a 10-fold buffer for T4 DNA polymerase (200 mM Tris-Cl, pH 8.4; 500 mM potassium chloride) is added to 20 μl (not more than 10 μg) of a solution of cDNA molecules, 1.5 μl 100 mM DTT solution, 3 μl of 1 mM deoxyribonucleotide solution and 1.0 μl (1.5 units) of T4 DNA polymerase and the final solution volume was adjusted to 30.0 μl using twice distilled water treated with DEPC. The solution is slowly mixed and incubated for 20 minutes at a temperature of 11 ° C. The reaction is stopped by the addition of an equal volume of a phenol-chloroform-isoamyl alcohol mixture (in a ratio of 25: 24: 1) and then the DNA which is contained in the aqueous phase is precipitated by the addition of sodium acetate and absolute ethyl alcohol solutions as described above. The resulting precipitate, which contains cDNA molecules, is dissolved in 10 μl of twice distilled water treated with DEPC. Oligonucleotide linkers phosphorylate by incubation with T4 polynucleotide kinase, immediately before use in the ligation reaction. To do this, collect the mixture by sequentially adding 10 μl of a solution of cDNA molecules (from the previous step), 2 μl of a 10-fold ligation buffer (10x = 200 mm Tris-Cl, pH 7.6; 50 mm potassium chloride), 2 μl 1 mM ATP solution, 2 μl (100 picomoles) of oligonucleotide linker solutions (5'-GCTTGAATTCAAGC-3 '/ 3'-CGAACTTAAGTTCG-5'), 2 μl 100 mM DTT solution, 2.0 μl ( 5 units) T4 - polynucleotide kinase. The solution is stirred and incubated for 5 minutes at a temperature of 37 ° C. Then add 6-8 units of T4 - DNA ligase and incubated for 6-20 hours at a temperature of 16 ° C.

Next, cDNA molecules are cleaved using 2 restriction enzymes - Hind III and EcoR1. For this, 10 μl of a 10-fold Hind III restriction endonuclease buffer and 10 μl (100 units) of Hind III restriction endonuclease (Invitrogen) are added to the cDNA solution, the volume of such a mixture is adjusted to 100 μl with twice distilled water. The resulting mixture was incubated for 2 hours at a temperature of 37 ° C. Then add 10 μl of a 10-fold buffer for restriction endonuclease Eco R1 and 10 μl (100 units) of restriction endonuclease Eco R1 (Invitrogen) and again incubated for 4 hours at 37 ° C. The reaction is stopped by the addition of an equal volume of a phenol-chloroform-isoamyl alcohol mixture (in a ratio of 25: 24: 1) and then the DNA which is contained in the aqueous phase is precipitated by the addition of sodium acetate and absolute ethyl alcohol solutions as described above.

Secondary separation of total RNA is carried out at the stage of fractionation and purification of the obtained cDNA molecules, for which gel filtration chromatography is used. A chromatographic column containing a specific size Sepharose particle (Novagen) was washed 5 times using 1 ml of neutralization buffer each time. A shared material, a cDNA population in a volume of 1 ml was applied and 25 μl fractions were collected in conical tubes.

Example 4. Cloning and creating multiple libraries.

The creation of libraries of all organs is carried out according to one model both for target cells and for cells that may be involved in the pathological process or capable of influencing its course, diagnosis and therapy.

To do this, use the DNA of the bacteriophage T7 (Novagen, T7 Select 10-3 vector), processed using 2 restriction endonucleases - Eco R1 and Hind III, i.e. the same type that was used to process the ends of the cDNA. The solution of the vector DNA molecules and the solution of cDNA molecules are mixed in a ratio of 1: 3, calculated on the mole of the substance, an equivalent amount of T4 - DNA ligase (GIBCO BRL) is added and incubated for no more than 28 hours at a temperature of 16 ° C. The reaction is stopped by the addition of an equal volume of a phenol-chloroform-isoamyl alcohol mixture (in a ratio of 25: 24: 1), and then the DNA which is contained in the aqueous phase is precipitated by the addition of sodium acetate and absolute ethyl alcohol solutions as described above.

The resulting recombinant cDNA molecules in an amount of not more than 1 μg are mixed with 250 μl of a solution of bacteriophage packaging proteins and incubated for 5 minutes at 0 ° C. A nutrient medium is then added and incubation is continued for another 60 minutes at a temperature of 37 ° C.

Example 5. Amplification of bacteriophages.

The bacteriophage T7 is cultured using E. coli strain BLT5403 as a host. In this case, amplification is carried out in several ways.

In one case (microamplification), a single plaque or eluate after screening, or some other small number of bacteriophages, is dissolved in 1 ml of culture medium prepared according to the Luria-Bertani method. Then add 1 ml of freshly prepared daily culture of the host strain with an optical density of 0.4-0.6 at a wavelength of 600 nm (OD ₆₀₀ = 0.4-0.6), which is prepared by building up 0.5 ml of night culture in 50 ml nutrient medium LB for 2-3 hours. A mixture of phages and bacteria with a total volume of 2 ml is incubated at a temperature of 37 ° C and 200 rpm on a shaker for 3-4 hours, observing complete lysis, which is expressed by the clarification of the contents of the tube.

In another case, macroamplification is carried out using flasks by inoculation of the required number of phages in 135 ml of freshly prepared daily culture of the host strain (Op ₆₀₀ = 0.4-0.6) and subsequent growth for 4-5 hours until complete lysis.

To the lysed cultures add 0.1 volume of 5 M solution of sodium chloride, intensively mix for 30 seconds and centrifuged for 3 minutes at 7000 * g and at a temperature of 4 ° C. The supernatant is transferred to a new tube and 1/6 of the volume of the solution containing 20% polyethylene glycol and 2.5 M sodium chloride is added, stirred vigorously, and incubated for at least 3 hours at 0 ° C. Next, the solution is centrifuged for 10 minutes at 8000 * g, and the residue containing phages is dissolved in 1 ml of standard TBS buffer (Tris - Borate Buffer) and the phage particles are again precipitated by adding a solution of polyethylene glycol - sodium chloride, followed by incubation and centrifugation for 10 minutes at 12000 * g and at a temperature of 4 ° C. The resulting precipitate was dissolved in 0.2 ml of TBS buffer.

Example 6. Screening (see figure 2).

Library screening is carried out both by repeated repetition of a one-step procedure, and using a multi-step complex procedure, which includes selection on combinations of various types of cells that may be involved in the pathological process or capable of influencing its course, diagnosis and therapy.

To do this, initiate a culture of vascular smooth muscle cells in Petri dishes with a diameter of 60 mm at the rate of 64,000 cells per cup and grow for 2 days. At the same time, cultures of endothelial cells, fibroblasts, HeLa, Hep-G2, in quantities of 48000, 24000, 16000, 16000 cells per cup, respectively, are initiated in similar plates and grown for 2 days.

Cells were washed intensively 5 times for 5 minutes, using 3 ml of HBSS buffer each time.

When using the one-step procedure, 10 ¹⁰ phage particles from any library are dissolved in 2 ml of HBSS buffer and incubated for 20 minutes with pre-incubation material (plastic). After that, the upper phase, which contains phages that did not bind to plastic, is transferred to previously washed vascular smooth muscle cells and incubated for 40 minutes at a temperature of + 4 ° С. After incubation, in order to remove unbound material, the cells are washed intensively 5 times for 5 minutes, using 3 ml of HBSS buffer each time, followed by elution of specific binding phages with 1 ml of elution buffer, which contains no more than 1, 0% sodium dodecyl sulfate, or with 1 ml of freshly prepared daily culture of the host strain. The titer of phages in the eluate is determined and the eluate is amplified to obtain at least 10 ¹⁰ phages that are incubated with a new portion of pre-incubation material and a new portion of vascular smooth muscle cells, repeating the selection cycle 3-5 times.

Using a multi-step procedure, 10 ¹⁰ phage particles from any library are dissolved in 2 ml of HBSS buffer and incubated for 20 minutes with pre-incubation material (a Petri dish in which instead of cells only medium was contained). After that, the upper phase, which contains phages not bound to plastic, is transferred to pre-washed endothelial cells and incubated for 40 minutes at a temperature of + 4 ° С, followed by transfer of the upper phase to a new portion of endothelial cells and similar incubation, which is repeated 2-3 times, and only after that the upper phase is directed to fibroblasts and similar selection is carried out 2-3 times. Or the upper phase after incubation on endothelial cells, without additional selection on fibroblasts, is transferred directly to vascular smooth muscle cells and incubated for 40 minutes at a temperature of + 4 ° C. Washing and elution are carried out as described above.

In another case, the upper phase after incubation with pre-incubation material (Petri dish, in which instead of the cells contained only medium) is transferred to previously washed HeLa cells and incubated with them for 40 minutes at a temperature of + 4 ° С, and then the upper phase is transferred to cells Hep-G2 and incubated with them for 40 minutes at a temperature of + 4 ° C, after which the upper phase is transferred to a new portion of HeLa cells and Hep-G2 cells, repeating the combined selection 2-3 times. The final upper phase is transferred to vascular smooth muscle cells and incubated for 40 minutes at a temperature of + 4 ° C.

In the next screening variant, the material after selection on immortalized cell types is transferred to endothelial cells and / or fibroblasts and only then to vascular smooth muscle cells.

Example 7. Performing combined testing of bacteriophages.

A. Enzyme-linked immunosorbent assay

The specificity of individual phage particles is assessed using them instead of the first antibody in a direct enzyme-linked immunosorbent assay, where target cells and cells that can be involved in the pathological process or can influence its course, diagnosis and therapy act as antigen.

To do this, all types of cells used are cultured in 96-well microplates, initiating a culture of 10,000 cells per well. Cells are grown for 2 days, while the medium is replaced every 24 hours, then the microplates are washed intensively 5 times for 5 minutes, each time using 200 μl of HBSS buffer. Then, 10 ⁶ -10 ⁷ phage particles with cells are incubated for 1 hour at 4 ° C with intensive subsequent washing with HBSS buffer and incubation with a second antibody for 1 hour at room temperature. A monoclonal antibody conjugated to horseradish peroxidase and recognizing a motif of 11 amino acids (Met-Ala-Ser-Met-Thr- (Gly) ₂ - (Gln) ₂ -Met-Gly) (Novagen, Cat. No. 69522) is used as the second antibody ), located at the N-terminus of protein No. 10, which is part of the surface of the T7 bacteriophage. The plates are then washed again with HBSS buffer and a second antibody is detected using a peroxidase substrate, the digestion of which is stopped by the addition of 50 μl of 1 M solution of sulfuric acid.

B. Binding Assay

The binding of individual phages is investigated using a single-step selection, in which the phage to be analyzed is tested in parallel for binding to target cells and cells that may be involved in the pathological process or can affect its course, diagnosis and therapy.

To do this, initiate a culture of SGMC cells in Petri dishes with a diameter of 60 mm, calculated at 64,000 cells per dish, and grown for 2 days. At the same time, cultures of endothelial cells, fibroblasts, HeLa, Hep-G2, in quantities of 48000, 24000, 16000, 16000 cells per cup, respectively, are initiated in similar plates and grown for 2 days. Use 10 ⁹ phage particles of individual phage, which are dissolved in 2 ml of HBSS buffer. Such a solution is first incubated for 20 minutes with pre-incubation material (a Petri dish in which instead of cells only medium was contained) and then the upper fraction is transferred to the test cells, previously washed 5 times with HBSS buffer, lasting 5 minutes each time. In this case, the procedure is simultaneously performed both for vascular smooth muscle cells (target cells) and for endothelial cells, fibroblasts, HeLa and Hep-G2 cells (cells that may be involved in the pathological process or are able to influence its course, diagnosis and therapy), using 2 temperature modes for all types of cells: + 4 ° C and + 37 ° C. After incubation, unbound particles were washed extensively using HBSS buffer, and then eluted using 1 ml of elution buffer. The titer of phages in the eluate is determined and the obtained data are compared for all types of cells. Moreover, the larger the ratio of the titer of phages obtained after elution with SGMK to the titer of phages obtained from other types of cells, the more specific the phage to SGMK and, accordingly, less specific to other types of cells.

B. Use of the polymerase chain reaction (PCR)

The specificity of individual phages is tested using PCR to identify clones, as well as to analyze their quantitative distribution when interacting with target cells.

To do this, initiate a culture of SGMC cells in Petri dishes with a diameter of 60 mm, calculated at 64,000 cells per dish, and grown for 2 days. At the same time, in similar plates, cultures of endothelial cells, fibroblasts, HeLa, Hep-G2 are initiated in quantities of 48000, 24000, 16000, 16000 cells per cup and grown for 2 days. Phage particles are mixed in equivalent amounts, typically 10 ⁷ particles of each type, based on a final solution volume of 2 ml. The resulting solution was first incubated for 20 minutes with pre-incubation material (a Petri dish in which instead of cells only medium was contained) and then the upper fraction was transferred to the test cells, previously washed 5 times with HBSS buffer, each lasting 5 minutes. In this case, the procedure is simultaneously performed both for vascular smooth muscle cells (target cells) and for endothelial cells, fibroblasts, HeLa and Hep-G2 cells (cells that may be involved in the pathological process or are able to influence its course, diagnosis and therapy), using 2 temperature modes for all types of cells: + 4 ° C and + 37 ° C. After incubation, unbound particles were washed extensively using HBSS buffer, after which the bound phages were eluted using 1 ml of elution buffer. Then determine the titer of phages in the eluate and scatter equal aliquots of the eluate to obtain a single plaque. 100 plaques were taken from each dish, distributed in groups of two, and DNA was isolated by lysis of phage particles. For this, phage particles are incubated in a 10 mM EDTA solution for 10 minutes at 65 ° C, followed by cooling and centrifugation for 3 minutes at 14000 * g. The resulting solution containing bacteriophage DNA is used as a template for PCR according to the standard scheme for this vector, directed by primers to the regions of the vector (T7 "Down" primer: 5'-AACCCCTCAAGACCCGTTTA-3 '; T7 "Up" primer: 5'-GGAGCTGTCGTATTCCAGTC -3 ') or specially designed primers. After that, the absolute frequency of occurrence of each phage is determined depending on the type of cell.

D. Stability and internalization test.

The stability of a specific phage, i.e. the ability to maintain its functional efficiency in the cell for as long as possible, to which it is specific and in the host organism itself, directly interferes with the ability to effectively penetrate the cell and maintain activity and specifically bombard intracellular targets.

To do this, initiate a culture of SGMC cells in Petri dishes with a diameter of 60 mm, calculated at 64,000 cells per dish, and grown for 2 days. At the same time, in similar plates, cultures of endothelial cells, fibroblasts, HeLa, Hep-G2 are initiated in quantities of 48000, 24000, 16000, 16000 cells per cup and grown for 2 days. 10 ¹⁰ phage particles representing the fraction obtained after phage elution as a result of 3 rounds of selection by multi-step screening are incubated with vascular smooth muscle cells at 2 temperature modes: + 4 ° C and + 37 ° C and for different periods of time. Next, the cells are washed and lysed for 10 minutes using 2 ml of lysis buffer (1 ml of 2% deoxycholic solution; 10 mM Tris; 2 mM EDTA, pH 8.0). After neutralization, the solution containing phages is scattered to single plaques, which are then selected to determine their nucleotide primary sequence, as well as to conduct additional testing.

Example 8. Determination of the nucleotide primary and encoded amino acid sequence and their computer analysis.

The DNA sequence of the vector genome region, representing the cDNA insert and limited by vector primers, is amplified using PCR and its nucleotide primary sequence is determined using an ABI Prism 310 automatic sequencer (Applied Biosystems Inc.), following the manufacturer's recommended reaction protocols.

Using specific recognition signals in the vector, the nucleotide primary sequence is translated into the amino acid sequence of the peptide. The nucleic acid sequence and the peptide sequence are checked for homology with the registered sequences in the databases of international agencies. To do this, use the computer analysis software package - GCG or a similar package for automatic sequence analysis, distributed by the US National Center for Biotechnological Information.

Claims (3)

1. A method for producing bacteriophages that specifically bind to target cells and intended for therapeutic purposes, comprising constructing a phage library of random peptides based on the oligonucleotide fragments encoding them, screening it to obtain bacteriophages that bind to target cells, and confirming the specificity of the selected bacteriophages, the fact that - oligonucleotide fragments encoding random peptides are obtained by the reverse transcription reaction using random primers and total RNA of target cells and other types of cells that may be involved in the pathological process or capable of influencing its diagnosis and therapy; - these oligonucleotide fragments are inserted into the bacteriophage vectors in the correct orientation and used to create phage libraries of random peptides for all types of cells used to isolate total RNA; - screening of the obtained phage libraries of random peptides is carried out in two stages, in the first of which bacteriophages capable of binding to target cells are selected, and in the second, non-binding to cells of other types used are selected from the bacteriophages selected in the first stage; - to confirm the specificity of bacteriophages to target cells, a combination of various tests is used. 2. The method according to claim 1, characterized in that vascular smooth muscle cells are selected as target cells. 3. The method according to claim 1, characterized in that as the bacteriophage vectors, vectors based on the DNA of the bacteriophage T7 are used.

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