RU2197987C2 - Carrier of foreign genetic information for gene therapy - Google Patents

Abstract

FIELD: medicine. SUBSTANCE: method deals with treating diseases associated with different genetic disorders obtained during patient's life or caused as a result of inherited pathology. The method deals with applying either lactoferrin (LF) and/or its conjugates as a carrier of foreign genetic information for gene therapy. At injecting laboratory animals with complexes of intact LF and plasmid DNA, containing different marker genes, genetic material penetrates tissues possessing membranous LF receptors and is expressed there. Moreover, synthesis of end products of introduced genes' expression is observed in different groups of muscular fibers. Expression of marker genes in different tissues takes place, also, at injecting complexes of DNA along with LF derivatives containing either DNA-condensing polycation or DNA-condensing polycation and high-molecular polymeric carrier - dextran or phycol. Efficiency of complex-formation with LF or with its derivatives and subsequent delivery of genetic material into cells depends upon plasmid nature. Advantage of the present innovation is in the fact that the mentioned product could be used in the field of gene therapy of different human and animal diseases. EFFECT: higher efficiency. 2 cl, 3 dwg

Description

 The invention relates to medicine, namely to the treatment of diseases associated with various genetic disorders acquired by a patient during his life or caused by a hereditary pathology.

Numerous means of delivering genetic material in the form of recombinant genes into eukaryotic cells are known. In this case, two fundamentally different groups of means are used:
1. Viral particles (packaging of target genes into viral particles) [1].

 2. Plasmids (bacterial autonomously replicating particles with integrated target genes) [2].

 When delivered using viruses, a very high efficiency of embedding target genes into recipient cells is achieved. However, in this case, the body can respond to the introduction of viral particles by an immune response and there is always a danger of the virus reversing to a pathogenic state. In addition, the production of viral particles with target genes is technologically very difficult. The use of plasmids as delivery vehicles has several advantages, since it does not cause immunization of the body, which allows plasmids to be reintroduced over a long period of time. This is especially important, for example, in the correction of genetic defects. However, the size of the exogenous gene inserted into the plasmid is not limited. In addition, the production of plasmids of a certain composition and properties is highly technological (rapid multiplication of plasmids in easily cultured bacterial cells). Therefore, the development of non-viral means of delivering genetic material to eukaryotic cells of an entire organism is highly relevant.

Non-viral methods can be divided into the following groups:
1. mechanical - direct introduction of material into cell nuclei (can only be used to introduce genetic material into cells outside the body) [3].

 2. physical - electroporation (the introduction of plasmids into cells under the influence of an electric field), the bombardment of tissues by microparticles [4].

 3. chemical (biological) - the use of liposomes [5], cationic lipids [6] and other compounds for DNA transfer.

 A disadvantage of non-viral methods for the correction of diseases associated with genetic disorders is the lack of tissue specificity of delivery of exogenous genetic material to the cells of the recipient. To achieve tissue-specific transport of polynucleotides, carriers of foreign genetic material are used, which include special components that ensure the transport of the target genetic material to target cells. For example, ligands of internalizing cell receptors. Such ligands can be: the plasma transport protein of blood plasma — transferrin [7], asialoglycoproteins or galactosyl derivatives of cationic polymers [8], as well as some other ligands.

 The objective of the invention is to develop an effective tissue-specific non-immunogenic means of delivery (carrier) of foreign genetic material into eukaryotic cells, which can be obtained with high reproducibility by a simple technological method.

 The task is realized by the use of lactoferrin (LF) as a carrier of foreign genetic information for gene therapy. Moreover, LF can be used as a carrier of foreign genetic information in the form of conjugates with DNA condensing agents and / or high molecular weight compounds.

LF is an iron-transporting protein of milk and mammalian neutrophils (structural homolog of plasma plasma transferrin) (9). This protein is found in large quantities in colostrum and breast milk. This protein consists of a single polypeptide chain with a molecular weight of ~ 80 kDa. The determination of the primary structure showed that the polypeptide chain consists of 680–700 amino acids and can be divided into 2 homologous parts, each of which binds 2 Fe ³⁺ ions very strongly [10]. To date, only the immunoprotective, antibacterial and / or anti-inflammatory properties of this protein are used [11, 12]. The previously described properties of LF do not explicitly imply the possibility of using it as a carrier of genetic information for gene therapy.

Examples of the invention:
For therapeutic purposes, we used complexes of the initial unmodified LF with the target DNA. The therapeutic effect was observed after transfection in the range from 2 to 60 days with standard laboratory animals. To identify the therapeutic effect, the degree of expression of the target gene was determined by the accumulation of the synthesized protein product [13] or by the specific activity of this product [14].

 Example 1

Before each introduction to 1 animal, 1-50 μg of plasmid DNA was mixed with 0.1 μg - 4 mg of purified starting LF (final concentration of LF in the sample 0.01-60 μg / μl) in a buffer containing 0.14 M NaCl and 0, 01 M NaHPO ₃ and in a volume of 5-100 μl.

 An analysis of the interaction of plasmid DNA with unmodified LF was carried out by electrophoresis in 0.8% agarose gel containing ethidium bromide [15]. The optimal ratios for the formation of a complex of DNA with LF were evaluated by a decrease in the electrophoretic mobility of DNA. FIG. 1 represents agarose electrophoresis of uncomplexed plasmid pDMDl (part A, lane 1), complexes of unmodified LF in increasing amounts, and plasmid pDMDl (part A, lanes 2-4).

 Example 2

 Transfection of muscle fibers in outbred mice with plasmids with the expressed bacterial beta-galactosidase gene using LF-based complexes.

 The bacterial betagalactosidase gene under the eukaryotic promoter in the plasmid pCMVLacZ was used as the reporter gene. Plasmid DNA constructs in complex with unmodified LF (for preparation see Example 1) were injected 1 to 5 times over 2 weeks into the quadriceps femoris muscle (musculus quadriceps femori). Under nembutal anesthesia, the animals were fixed on a special table, the skin above the muscle was cut, and under the control of a binocular magnifier, 5-100 μl of a solution containing 1 test compounds or an uncomplexed plasmid was introduced as a control using an insulin syringe. Animals were slaughtered 2-60 days after administration of the complexes. After histochemical staining for the detection of betagalactosidase activity to establish the nature and localization of staining, the muscles treated with X-gal were paraffin embedded in a standard procedure and histological preparations were prepared [16]. Plots of tissue with betagalactosidase activity were detected by the appearance of specific blue-green staining.

 As a result, it was obtained that in the muscle of the introduction of DNA complexes with intact LF, strands corresponding to transfected fibers are clearly stained. Figure 2 presents the results of cytochemical detection of beta-galactosidase gene activity on histological sections of musculus quadriceps femori in the contralateral muscle (A), in the injection muscle (B) after intramuscular administration to the mice of the plasmid pCMVLacZ in combination with unmodified LF. An analysis of the nature and nature of the color obtained on paraffin sections confirmed the correspondence of the stained areas to muscle fibers. In the same muscle of the contralateral limb and in other muscles of the anterior and posterior limbs, color was not observed. In control experiments, no stained muscle fibers containing active beta-galactosidase, the product of the expression of the marker exogenous gene, were detected.

 Thus, complexes based on intact LF and plasmid DNA penetrated muscle fibers and ensured the expression of exogenous genes contained in the plasmid.

 Example 3

 Transfection of muscle fibers of mdx mice (Duchenne model of myodystrophy) with a recombinant plasmid containing the human dystrophin gene using intact human LF.

 The study was performed on male mice of F1 CBAxC57BL110J + / + hybrids and on C57BL6J mdx / mdx (biological models of Duchenne myodystrophy) at the age of about 3 months and weighing up to 25 g. A cDNA copy (12 bp) was used as a marker the human dystrophin gene in plasmid pDMDl (16 kb) (pJ4Ω vector with the dystrophin gene expressed in eukaryotic cells under the control of the MoMuLTR promoter). LF-plasmid DNA complexes were prepared and administered to animals as described in Example 1.

5-60 days after administration, the mice were sacrificed by dislocation of the cervical vertebrae, the quadriceps muscles of the experimental and contralateral limbs, as well as other muscles were removed, fixed in liquid nitrogen and serial cryostatic sections 5-10 μm thick were prepared. Immunocytochemical staining for the presence of dystrophin was carried out by incubating sections with rabbit polyclonal antibodies to the C-terminal portion of human dystrophin and then in a medium with sheep anti-rabbit antibodies containing a fluorescent label. Dystrophin-positive muscle fibers were counted using fluorescence microscopes from Leitz and Axioscope (Opton) at a magnification of 240-1200. After recording the number of dystrophin-positive fibers, the sections were washed and stained with hematoxylin-eosin. The average number of fibers per section was counted. On transverse cryosections of muscle fibers stained with dystrophin-specific antibodies, clusters of dystrophin-positive muscle fibers were observed that were not found in the muscles of mdx mice. FIG. Figure 3 presents the result of immunochemical staining of cryostatic sections of musculus quadriceps femori for dystrophy: mdx mouse on day 21 after intramuscular transfection with pDMDl + complex unmodified LF (A), intact mdx mouse (B), CBAxC57B110J + / + mouse - positive control (C). A characteristic feature of dystrophin gene cDNA transfection was a distinct polymorphism of localization of human dystrophin in mouse muscle fibers. 3 main variants of the intracellular distribution of dystrophin were identified:
1) typical clusters of dystrophin-positive muscle fibers with a clear subarcolemal localization of dystrophin;
2) fibers with fragments of a priscarolem dystrophin;
3) fibers with diffuse coloration of myoplasma on dystrophy.

 By visual assessment, most dystrophin-positive fibers belong to the first two types.

 The introduction of an uncomplexed ("naked") plasmid pDMDl leads to the appearance of from 2.1 to 3.3% of dystrophin-positive muscle fibers. The introduction of complexes of LF with pDMDl, depending on the duration of exposure, induces the appearance of from 5.5 to 14.4% of dystrophin-positive muscle fibers. At the same time, an increase in the number of dystrophin-positive muscle fibers was observed both in the introduction muscle, and in the same muscle of the contralateral paw and in the muscles of other extremities. According to the number of dystrophin-positive muscle fibers, muscles of different extremities did not differ significantly, however, the maximum effect was observed in the muscles of the contralateral paw.

 Thus, the ability of LF to stimulate the penetration and expression of the human dystrophin gene in dystrophin-free muscle fibers of mutant mice is clearly manifested.

 For therapeutic purposes, we also used complexes of target DNA with various derivatives of LF, for example, LF conjugates with DNA condensing polycations (polylysine, polyethyleneimine, etc.), high molecular weight polymers (ficoll, dextran, etc.) and other compounds that provide special functions of the final complexes . The therapeutic effect was observed after transfection in the range from 2 to 60 days with standard laboratory animals. To identify the therapeutic effect, the degree of expression of the target gene was determined by the accumulation of the synthesized protein product [13] or by the specific activity of this product [14].

 Protein-polynucleotide particles, including LF conjugates, were prepared sequentially. First, various LF conjugates were synthesized, which were then bound under certain conditions to the target gene.

 Example 4

 The LF – polylysine-based conjugates were synthesized by adding 1–50-fold molar excess of iodoacetic anhydride to the purified LF, the mixture was incubated with constant stirring for 1 h at room temperature, the excess of low molecular weight reagent was separated by dialysis. Similarly, iodoacetic acid anhydride was treated with polylysine (mol. M 2 - 150 kDa). Then, a 50-100-fold (relative to the initial amount of iodoacetic anhydride) excess dithiothreitol was added to the activated polylysine, and the mixture was dialyzed against physiological saline after an hour incubation at room temperature. Polylysine and LF modified in this way were mixed in a quantitative ratio of 1: 1 - 1:15, respectively.

 Example 5

The synthesis of conjugates based on the carrier ficol-400 was carried out according to the scheme: 50-500 mg of ficol-400 was dissolved in 3-30 ml of 50% aqueous acetone and 0.15-1.5 ml of BrCN acetone solution (100 mg / ml) and 0 were added 15-1.5 ml of an acetone solution of triethylamine (150 mg / ml). The mixture was incubated for 5 min at a temperature of -20 ^o C. Then the activated ficol precipitated with acetone with the addition of 0.2 N. Hcl and washed 1-5 times. Then, a 10-100-fold molar excess of reduced glutathione was added to an aliquot of the ficol dissolved in water, and the pH of the solution was adjusted to 7.5-9.4 with NaHCO ₃ - Na ₂ CO ₃ . The mixture was incubated for 12 hours at room temperature. Excess unreacted reagent was separated by ultrafiltration. A 1-15-fold molar excess of polylysine modified with iodoacetate and a 1-50-fold molar excess of LF also modified with iodoacetate were added to the thiol derivative of ficol.

 To prove the production of conjugates, the method of polyacrylamide gel electrophoresis under denaturing conditions was used [17]. The change in the electrophoretic mobility of the test sample, in contrast to the electrophoretic mobility of unmodified LF, confirmed the formation of a chemical bond. At the same time, immunological characterization of the conjugates was carried out by the method of missile immunophoresis [18], immunoblotting [19]. The proposed method for the synthesis of derivatives of LF really makes it possible to obtain particles that include both LF and additional special-purpose components (polycations, ficol, etc.).

 Example 6

The resulting conjugates were combined with plasmid DNA by gradually adding the conjugate solution to the DNA solution at a temperature of 0 - + 4 ^o With constant stirring and the content of NaCl in solutions in the amount of 0.15-1.5 M.

 An analysis of the interaction of plasmid DNA with LF conjugates was carried out by electrophoresis in a 0.8% agarose gel containing ethidium bromide. Increasing amounts of conjugate (Example 4 or 5) were added to 1 μg of DNA in a volume equal to the volume of the DNA solution. After a 15-min incubation at room temperature, the samples were subjected to electrophoresis in a Tris-borate system, pH 8.0 [15]. The optimal ratios for the formation of a complex of DNA with LF and with conjugates were evaluated by a decrease in the electrophoretic mobility of DNA. FIG. 1 represents agarose electrophoresis of uncomplexed plasmid pCMVLacZ (part B, lane 1), complexes of the LF conjugate, made as shown in example 4, in increasing quantities, and plasmid pCMVLacZ (part B, lanes 2-5).

 Example 7

 Transfection of muscle fibers of mdx mice (Duchenne myodystrophy model) with a recombinant plasmid containing the human dystrophin gene using human LF conjugates.

 Protein-polynucleotide particles comprising LF conjugates were prepared as described in Example 6.

 Dystrophin gene expression in the muscle fibers of mutant mice was evaluated as described in Example 2. It was found that LF derivatives, as well as the original LF, stimulate dystrophin production in muscle fibers with the appearance of up to 6.8% dystrophin-positive muscle fibers.

 The selection of the optimal tissue-specific carrier (unmodified LF or conjugates based on it) of foreign genetic information is established separately in each case, which is associated with the nature of the nucleotide sequence used.

Distinctive features and advantages of gene-therapeutic complexes based on the LF carrier or its conjugates are associated with the following properties of the protein:
1. Resistance of the fragment binding to its own LF receptors to proteolytic degradation by trypsin-like enzymes.

 2. Resistance to lysosomal enzymes.

 3. The presence of a nuclear translocation signal (provides transport of LF-containing complexes directly to the cell nucleus).

 4. The absence of an immune response when introduced to individuals of the same species as the source of this protein. Therefore, the introduction of complexes based on LF can be repeated, which is important in the treatment of, for example, hereditary diseases, when a constant, throughout life, correction of impaired gene function is necessary.

 5. The low initial concentration of LF in the blood plasma does not cause competition between free LF of the body and the introduced DNA complexes with LF. However, LF can be easily obtained from milk or as a recombinant protein in a bacterial culture.

List of figures
Figure 1. Agarose gel electrophoresis of plasmid DNA complexes with unmodified LF (part A) and with LF conjugate (part B).

 FIG. 2. Cytochemical detection of beta-galactosidase gene activity on histological sections of musculus quadriceps femori in the contralateral muscle (A), in the injection muscle (B) after intramuscular administration to the mice of the plasmid pCMVLacZ in combination with unmodified LF.

 FIG. 3. Immunochemical staining of cryostat sections of musculus quadriceps femori for dystrophy: mdx mouse 21 days after intramuscular transfection with pDMD 1 + complex unmodified LF (A), intact mdx mouse, CBAxC57B110J + / + mouse - positive control (B).

Sources of information
1. Biotechnology, 1995, 13, pp. 222-225.

 2. J. Mol. Med., 1995, 73, 10, pp. 479-486.

 3. Cell, 1980 22, (2 Pt 2), pp. 479-488.

 4. Science, 1990, 250, pp. 1421-1423.

 5. J. Biol. Chem., 1980, 255, pp. 10431-10435.

 6. Hum. Gene Therapy, 1996, 7, pp. 1437-1446.

 7. Proc. Natl. Acad Sci. USA, 1991, v. 88, pp. 8850-8854.

 8. Bioconjugate Chem., 1995, 6, pp. 401-410.

 9. Acta Chem. Scand., 1960, 14, pp. 510-512.

 10. Proc. Natl. Acad Sci. USA, 1987, 84, pp. 1769-1763.

 11. The application for a patent of the Russian Federation 94035529, published on 09/20/96.

 12. The application for a patent of the Russian Federation 95102930/14, published on 08.27.97.

 13. Genetics, 1998, v. 34, 7, p. 876-882.

 14. Cell, 1984 (part 2), 39, pp. 653-662.

 15. Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Lab., Cold Spring Harbor, NY), 1989. V. 3.

 16. Microscopic technology. M .: Soviet science, 1957, 137 p.

 17. Nature, 1970, 227, pp. 680-685.

 18. Anal. Bioch, 1966, 15, 1, pp. 45-52.

 19. Anal. Biochem., 1981, 112, pp. 195-203.

Claims (2)

 1. The use of lactoferrin as a carrier of foreign genetic information for gene therapy.  2. A carrier of foreign genetic information, which is a conjugate of lactoferrin with DNA condensing polycations or with DNA condensing polycations and ficol or dextran.

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