RU2190018C2 - Molecular vector for delivering genes to target cells - Google Patents

Abstract

FIELD: medicine; biological engineering. SUBSTANCE: molecular structure is a set of particles with recombinant DNA and gene to be delivered being disposed in its center and antibodies to target cells being spaced over its surface. The antibodies make part of spermidinepolyglucin-conjugate capable of retaining the conjugate on the negatively charged DNA owing to positive charge of spermidine taking place and of stimulating DNA molecules to penetrate into eukaryotic cells. EFFECT: enhanced effectiveness in producing means for delivering genes to target cells. 4 dwg, 6 tbl

Description

 The invention relates to the field of biotechnology, medicine, immunology; it can be used in the pharmaceutical industry and molecular pharmacology and is a method for targeted gene delivery to target cells.

 Advances in genetics, molecular biology and biotechnology determine the development of strategies and tactics for introducing DNA into cells for both gene therapy and the development of DNA vaccines. In the early 1990s, WHO compiled and approved recommendations for work in these areas. In the decision of the eighth session of the general meeting of the Russian Academy of Medical Sciences in terms of the development of priority areas of basic medical research, genetic diagnostics and gene therapy are in the first place [1]. Much attention was paid to the creation and development of DNA vaccines at the 1998 Vaccines and Immunization international conference [2]. Both created drugs and their mechanisms of action were discussed.

 Of the main problems associated with the safety of these methods, two are the main ones: the possible mutagenic effect of DNA embedded in the human cell genome, and immunopathology, which can occur during prolonged production of antigen in the body [3]. These problems are solved both by creating special recombinant vectors containing genes, and by developing methods for delivering genes to cells. Today, one of the promising and researched areas is the development of methods for targeted gene delivery to target cells of the body. The bottom line is to deliver the gene precisely to those cells in which it must function.

 Known methods for creating recombinant vectors and their introduction into the body [4]. Initially, “unprotected” recombinant DNAs were introduced, which were rapidly inactivated by blood nucleases and the efficiency of DNA delivery to cells was low [5-7].

 Currently, methods are developed for the protection of introduced DNA (liposomes, etc.) and methods for the delivery of DNA to target cells and penetration into them. So in [8] (prototype) describes the use of a complex of protein with polycationic nucleic acids. The authors used transferritin as a protein and showed that the complex interacts with transferritin receptors, penetrates cells through pinocytosis, and DNA is expressed in cells. The disadvantage of this method is the lack of universality of DNA delivery (to other target cells) and the relatively low efficiency of DNA penetration into cells.

 An object of the invention is the creation of a highly efficient molecular vector as a means of targeted gene delivery to target cells. For gene therapy and DNA vaccination of people, it seems promising to create such a vector construct that would protect a recombinant plasmid with an integrated gene from the action of blood nucleases, and, at the same time, would have a stimulator for the penetration of DNA into cells. The following are used as stimulators of DNA penetration into eukaryotic cells: DEAE-dextran, calcium phosphate, and some others [9].

 The problem is solved by creating a molecular vector - a construct that is virus-like particles, in the center of which is a recombinant plasmid DNA with a delivered gene, and on the surface of the antibody to target cells. Antibodies are part of the conjugate: spermidine-polyglucin, which has two functions: it holds the conjugate on negatively charged DNA due to the positive charge of spermidine and at the same time is an analogue of DEAE-dextran, a stimulator of the penetration of DNA molecules into eukaryotic cells.

 The invention consists in the following.

 The molecular vector for delivering genes to target cells is a particle in the center of which is a recombinant plasmid DNA containing the delivered genes, and antibodies to the target cells on the surface.

 To assemble the molecular vector, a recombinant plasmid DNA containing the delivered gene is created, and it is purified by known methods [10]. Antibodies to target cells are obtained using standard methods of immunization with antigens (target cells or their components), followed by purification of antibodies from blood serum [11].

The plasmid DNA with the delivered gene is coated with a conjugate layer (FIG. 1):
- polyglucin with a molecular weight of about 60,000 D is activated with sodium periodate, which is then removed by gel filtration on a Sephadex G-50 column in potassium phosphate buffer;
- spermidine and antibodies are added to activated polyglucin in the ratio of 100 spermidine molecules and 1 antibody to 10 polyglucin molecules;
- after incubation, unbound components are removed by gel filtration on Sephadex G-50 in buffer: 0.1 M Tris-HCl, pH 8.3;
- the resulting conjugate is added in excess to plasmid DNA and, after incubation, the conjugate is removed from excess by gel filtration on a CL-6B Sepharose column in physiological saline.

 The process of obtaining a molecular vector for gene delivery to target cells is presented in figure 2. Stability of the plasmid in cells and gene expression is determined by known methods (mapping of plasmid DNA with restriction enzymes, ELISA, PCR, etc.).

 New features, compared with the known constructions, are: the use of a molecular vector for gene delivery containing recombinant plasmid DNA with the delivered gene in the center, and on the antibody surface to target cells. Antibodies are retained on the surface due to their incorporation into the conjugate: spermidine-polyglucin, which has two functions: it holds the conjugate on negatively charged DNA due to the positive charge of spermidine and at the same time is an analogue of DEAE-dextran, a stimulator of the penetration of DNA molecules into eukaryotic cells .

 It is this combination of features that provides targeted delivery of ions to target cells and a higher degree of transformation of recombinant DNA cells. According to the proposed scheme, a molecular vector design was created, which is a virus-like particle, in the center of which there is a recombinant plasmid DNA with a delivered gene, and on the surface - antibodies to target cells and its biological activity is checked. It was shown that the introduced gene is delivered and accumulates in those cells of the body for which antibodies are located on the surface of the structure. Thus, we can conclude that the proposed technical solution allows targeted delivery of genes to target cells, protects them from the action of blood nucleases and, in addition, the polyglyukin-spermidine complex, which is an analogue of DEAE-dextran used in vitro to stimulate the transformation of eukaryotic cells, similarly facilitates the penetration of delivered genes into cells.

The list of graphic materials:
figure 1. Molecular vector for gene delivery to target cells:
1 - plasmid DNA containing the delivered gene; 2 - conjugate: spermidine-polyglucin - antibodies to target cells.

figure 2. Gel filtration on a column with sepharose CL-6B:
1 - plasmid DNA, 2 - the same, after incubation with the conjugate: spermndine-polyglucin - antibodies.

 FIG. 3. Restriction analysis (Hinf I) of plasmid DNA. Pathways of agarose gel electrophoregrams: 1 - initial plasmid DNA, 2 - plasmid DNA after passage in lymphocytes, 3 - the same, in keratinocytes. H - DNA plasmid pBR322 + Hinf I.

 FIG. 4. Analysis of lymphocyte proteins obtained on the 10th day after immunization of mice with a molecular vector. A - electrophoregram in a 15% polycrylamide gel, B - immunoblotting using polyclonal anti-α-TNF antibodies, C - the same, using polyclonal anti-γ-IFN antibodies.

 The invention is illustrated by the following examples of specific performance.

 Example 1. Plasmids for gene delivery.

 A plasmid containing the gene encoding the biosynthesis of the hybrid protein interferon-gamma - tumor necrosis factor-alpha is used [12].

 Example 2. Obtaining and purification of antibodies to target cells.

Antibodies are obtained in a standard manner [11]. To do this, murine lymphocytes are isolated, and they are purified by centrifugation in a gradient of hypakicoll. Antigens, 10 ⁵ cells in 0.1 ml of physical. solution, administered three times intraperitoneally with an interval of two weeks. Blood sampling is carried out 10 days after the last immunization. Ammonium sulfate fractionation followed by chromatography on a DEAE cellulose column is used to purify antibodies from sera. As antibodies to human lymphocytes, a pharmacy preparation of anti-lymphocyte immunoglobulin (equine against human lymphocytes) is used, additionally purified on a DEAE cellulose column.

 Example 3. The creation of a molecular vector for gene delivery to target cells.

 At the center of the vector is an intact plasmid DNA containing the delivered gene, and on the surface are antibodies to target cells or proteins having affinity for receptors of target cells. For communication, a polyglucin conjugate with spermidine and an antibody or protein is used. A positively charged spermidine binds the conjugate to plasmid DNA. Schematic view of the structure shown in figure 1.

 To obtain a polysaccharide insert, 50 mg (0.8 μM) of polyglucin (60,000 D) are treated with a 0.5 M sodium periodate solution for 20-30 min and then gel filtration is performed on a Sephadex G-50 column in 20 mM phosphate buffer ( pH 7.6). After that, 1.3 mg of spermidine (8 μM) and 22 mg (0.17 μM) of antibodies or proteins are added to the activated polyglucin solution. The mixture was incubated overnight, then sodium borohydride was added, stirred for another 2 hours, and unreacted components were removed by gel filtration on Sephadex G-50 using 100 mM Tris-HCl (pH 8.3). Thus obtained preparation contains 23.2 mg of protein. Since the initial components (spermidine and antibodies) are introduced in a certain ratio, it can be assumed that the resulting conjugate contains them in the same ratio, i.e. about 1: 10: 0.1.

To assemble the construct, a conjugate (200 µg protein) is added to a solution containing 400 μg of nucleotide material (about 0.12 nM) in physiological saline, the mixture is incubated for 2 hours at 4 ^° C, and the resulting structure is separated from unbound components by gel filtration on Sepharose CL-6B (Fig. 2).

 Investigation of the properties of molecular construction.

 The final constructs contained approximately 80 μg of protein (about 0.6 nM antibodies with a molar mass of about 130,000 D per 0.12 nM plasmid). Thus, they had an approximate ratio: for 1 molecule of the plasmid - 50 molecules of the polysaccharide insert - 5 molecules of antibodies. They say. a mass of about 6,700,000 D, and when gel chromatography on a CL-6B Sepharose column, they eluted in free volume (FIG. 2).

 From theoretical calculations on the surface of a supercoiled plasmid molecule, mol. weighing about 3000000-4000000 D of a spherical shape can fit about 50-60 polyglucin molecules (mol. mass of 60,000 D). Since in the experiment this ratio was 1:50, it can be assumed that the DNA material is completely “packed”.

To check the completeness of the packaging of the nucleotide material in the assembled construct, it was processed with a mixture of DNase and RNase (10 μg / ml). Experience has shown that while for the initial plasmid DNA complete degradation was observed after 30 min of incubation, the nucleotide material remained intact for 24 hours in the assembled construct. When storing the structure in sterile conditions at a temperature of 4 ^o C, its properties did not change for at least 1 month.

 Example 4. Biological tests of molecular construction.

 A) In vitro tests Initially, studies on the delivery of plasmid DNA, its stability, and its ability to penetrate cells were carried out in in vitro experiments. In this case, the following cells were used: lymphocytes, macrophages, hepatocytes, cardiomyocytes, fibroblasts, brain cells, and lungs. The cells of two animal species were used: mouse and 15-day-old chicken embryos.

To 0.3 ml of cell suspensions with a concentration of 10 ⁶ cells / ml, 5 μg of pure plasmid preparations and 5 μg of "packed" in a construct with two types of antibodies on the surface were added. The first - rabbit antibodies against murine lymphocytes, the second - anti-lymphocyte immunoglobulin (equine against human lymphocytes). After various incubation time periods, cells were harvested by centrifugation and analyzed for the presence of plasmid material as described above. E. coli cells grew on plates with ampicillin only after contact with nucleotide material obtained from lymphocytes, macrophages and culture medium using a molecular vector. The data are presented in Tables 1, 2. When using pure plasmid for the introduction of the E. coli cell growth gene on a selective medium, no observation was made; only a few colonies from the culture medium grew after 30 minutes of incubation.

 As can be seen from tables 1 and 2, if in the first 3 hours the accumulation of plasmids in lymphocytes and macrophages proceeds at approximately the same rate, then in the lymphocytes there is more plasmid material. By 72 hours of incubation in macrophages of the plasmid material, about 35% of the maximum value remains, while its level in lymphocytes remains. The tables also show that the dynamics of the accumulation of plasmid material in cells using molecular vectors with antibodies against lymphocytes obtained from different sources correlate. Similar data were obtained using chicken embryo cell cultures for incubation with plasmid material.

 Similar experiments were carried out with a molecular vector containing guinea pig antibodies to mouse keratinocytes on the surface. When using a pure plasmid to introduce the E. coli cell growth gene on selective medium, as in previous experiments, was not observed. But growth of transformed E. coli cells was observed after incubation with nucleotide material derived from fibroblasts. The data are presented in table 3.

 B) In vivo tests. Animals were injected intraperitoneally with 10 μg, both pure plasmid preparation, and 10 μg in the constructs. At certain time intervals, the animals were sacrificed with a cervical dislocation, organs were removed, they were washed from blood residues, tissue cells were homogenized, macrophage and lymphocyte cells were separated by the methods described in [11]. Nucleotide material was extracted from cell cultures as described above and analyzed for the presence of a plasmid. When mice were immunized with preparations of a pure E. coli cell growth plasmid on selective medium, they were not subsequently observed. The distribution of the plasmid material upon immunization with the preparations of a molecular vector with antibodies against lymphocytes on its surface is presented in table 4.

 No plasmid material was found in the cells of the heart, muscles, brain, skin integument. The presence of plasmids in the organs shown in table 4 can probably be explained by the presence of lymphocytes and macrophages. The distribution of plasmid material among blood components was investigated. The results are presented in table 5.

 From table 5 it is seen that the plasmid material accumulates and is deposited mainly in the cells of the lymphocytes. Its detection in macrophages is explained by phagocytosis, and a decrease in its level in macrophages is due to the corresponding killing of material that has entered the cells. The decrease in the level of plasmid material in the fraction of blood lymphocytes by the 60th and 90th days can be explained by the deposition of lymphocytes in the spleen and lymph nodes, as well as the elimination of part of the lymphocytes from the body when they are renewed. The data in table 4 show that the accumulation of plasmid material occurs in the spleen, the place of deposition of lymphocytes.

 The dynamics of the distribution of plasmid material when using a molecular vector with antibodies to keratinocytes on its surface in the blood of mice is similar to that shown in table 3. The distribution of plasmid material in this case by organs is presented in table 6.

 From table 6 it is seen that the plasmid material during the experiment accumulates in the skin and by the 60th, 90th day its level flows.

Example 5. Determination of the stability of the plasmid with the delivered gene after transformation of eukaryotic cells
For the expression of the delivered gene in eukaryotic cells, the stability of the plasmid itself carrying the gene plays a significant role. It is known that foreign genetic material in cells can be eliminated (removed) or subjected to degradation, including with the preservation of “markers”, but with the loss of the delivered gene (deletion). Since the plasmid material isolated from eukaryotic cells allowed the E. coli cells transformed by it to grow on selective medium, we can conclude that the plasmid is present and the marker is preserved. To verify the segregation stability of plasmids, they were physically mapped (restriction analysis). In FIG. Figure 3 shows the electrophoresis of hydrolysis of plasmid samples isolated from E. coli cells transformed with nucleotide material obtained from lymphocytes and fibroblasts during in vivo tests. Direct restriction analysis of plasmid samples isolated from eukaryotic cells was difficult, possibly due to partial methylation of DNA. As can be seen from FIG. 3, plasmid DNA samples are hydrolyzed by the finely chopped restriction endonuclease Hinf I to the same number of fragments, the sizes of which coincide with those for the fragments of the original plasmid DNA. Thus, we can conclude that the plasmid DNA used for experiments in eukaryotic cells is quite stable
Example 6. Determination of expression of delivered genes in cells
Since the gene used in the experiment was integrated under the control of prokaryotic promoters, we did not particularly expect its expression. Nevertheless, when setting up an immunoblot with antibodies to TNF-α and γ-interferon, it was found that in the lymphocytes obtained from mice on the 10th day after the introduction of the molecular construct on the immunoblot, a band with mol. weighing about 32,000 D, interacting with antibodies against both TNF and against interferon (Fig. 4). In the study of serum, the corresponding bands were not visually detected. Thus, the use of the proposed molecular vector for gene delivery to target cells allows you to:
- purposefully deliver genes to target cells, minimizing their entry into other types of cells;
- use the genes of infectious agents in recombinant plasmid DNA (the "central core" of the particles) as a DNA vaccine;
- use a positively charged polyglucin-spermidine complex (analogue of DEAE-dextran) as a stimulator of DNA penetration into cells.

Literature
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Claims (1)

 A molecular vector for delivering a gene to target cells, which is a molecular construct containing a plasmid in the center, including a gene delivered to the target cells coated with a conjugate: spermidine-polyglucin - antibodies to target cells.

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