RU2138553C1 - Transfection composition for higher eucaryotic cells, nucleic acid complex useful as component of transfection composition, conjugate useful as component of transfection composition, endosomolytic peptide useful as component of transfection composition - Google Patents

Abstract

FIELD: biotechnology. SUBSTANCE: invention relates to the transfection composition containing the nucleic acid complex, substances exhibiting an affinity to nucleic acid and an endosomolytic peptide. Invention can be used for transfer of nucleic acids in higher eucaryotic cells. EFFECT: alleviated process of introduction of nucleic acids in cells. 75 cl, 1 tbl, 41 ex

Description

 The invention relates to genetic engineering, and more particularly to a transfection composition for higher eukaryotic cells, a nucleic acid complex and an endosomolytic peptide as components of the composition, a conjugate as a component of the complex.

 Particularly in gene therapy, an effective system is required for introducing nucleic acid into living cells. Genes are transferred into cells in order to achieve the synthesis of therapeutic active genetic products in a living organism, for example, in order to replace a defective gene in the event of a genetic defect. "Standard" gene therapy is based on the principle of achieving long-term treatment by a single dose. However, there is also a need for treatment methods in which therapeutically active DNA (or mRNA) is given as a medicine ("therapeutic gene agent") once or, if necessary, repeatedly. Examples of genetically caused diseases in which gene therapy provides a promising approach are hemophilia, β-thallasemia and "serous combined immune weakness", a syndrome caused by a genetically induced lack of the adenosine deaminase enzyme. Another area of application is immune regulation, in which humoral or intracellular immunity is provided by the production of a functional nucleic acid encoding a secreted protein antigen or a non-secreted protein antigen, which can be considered as vaccination. Other examples of genetic defects in which you can give a nucleic acid encoding a defective gene, for example, in a form that is specific for each case, include muscular dystrophy (dystrophin gene), fibrocystic degeneration (regulatory transmembrane conductivity gene of viscidosis), hyperolestirolemia (receptor gene low density lipoprotein). Gene therapy also includes a method according to which hormones, growth factors or proteins of cytotoxic or modulating immune system activity should be synthesized in the body.

 Gene therapy is also seen as a promising method for treating cancer by giving the so-called "cancer vaccines". To increase the immunogenicity of tumor cells, cancer vaccines are either given greater antigenicity or the ability to produce certain substances that can modulate the immune system, such as cytokines, in order to elicit an immune response. This method is carried out by transfection of DNA cells encoding a cytokine, for example, IL-2, IL-4, γ-interferon, α-factor of tumor necrosis. Until now, the introduction of a gene into autologous tumor cells has been via retroviral vectors.

 Known transfection composition for hepatocytes, which is a conjugate of a polycation having the ability to modify, for example, polylisin, and a glycoprotein with the ability to modify, and the conjugate binds to receptors on the surface of hepatocytes (see G.I. Wu, S.N. Wu, J. Biol. Chem. 263, 29, pp. 14621-14624, 1988).

 A disadvantage of the known composition is that it can only be used to transfer nucleic acids to hepatocytes. Therefore, the operational capabilities of the known composition are limited.

 The objective of the invention is to expand the operational capabilities of transfection systems using a composition that allows the transfer of nucleic acids into higher eukaryotic cells.

 The term "transfer" is understood within the framework of the present invention not only to introduce nucleic acid complexes into the cell through the cell membrane, but also to transport the complexes or nucleic acid released from them inside the cell until a suitable site is to be expressed. Higher eukaryotic cells are well known. They do not include yeast.

 Many viruses enter the eukaryotic host based on mechanisms that, in principle, correspond to the mechanism of endocytosis carried out with the participation of the receptor. A viral infection based on this mechanism usually begins with particles of the virus binding to receptors on the cell membrane, after which the virus enters the cell. The process of entering the cell inside is carried out in the same way as the entry of physiological ligands or macromolecules into the cell. First, receptors on the cell surface classify themselves into groups and the membrane is inverted inward and forms a vesicle equipped with a mesh coating. After the vesicle is freed from the mesh coating, oxidation takes place in it with the help of a propon pump in the membrane. This leads to the separation of the virus from the nucleolus. Depending on whether the virus is lipid-coated or not, two types of separation of the virus from the nucleolus are possible. In the case of so-called “naked” viruses (such as, for example, adenovirus, poliovirus, rhinovirus), it was believed that a low pH value causes a change in the configuration of the virus proteins, which leads to the separation of hydrophobic domains that are not accessible at a physiological pH value. Thus, these domains acquire the ability to interact with the nucleolus membrane and thereby determine the separation of the virus genome from the nucleolus and its entry into the cytoplasm. For lipid-coated viruses (such as, for example, vesicular stomatitis viruses, Semliki Forest virus, influenza virus), it is assumed that a low pH modifies the structure or configuration of some proteins of the virus, resulting in a fusion of the virus membrane with the nucleolus membrane. Viruses that penetrate the cell using this mechanism have certain molecular abilities that give them the ability to destroy the nucleolus membrane in order to enter the cytoplasm.

 Other viruses, such as coated Sendai viruses, AIDS virus and some strains of Moloney's leukemia virus, or uncoated SV40 viruses and polyomas, do not require a low pH to penetrate the cell. Either they can cause fusion with the membrane directly on the surface of the cell (Sendai virus, possibly the AIDS virus), or they are capable of implementing mechanisms to decompose the cell membrane or pass through it. It is contemplated that viruses that are independent of a particular pH may also use the mechanism of endocytosis. The starting point for solving the problem of the invention was the use of a mechanism by which some viruses penetrate eukaryotic cells in order to achieve efficient transfer of nucleic acid complexes into the cell and thereby increase expression.

 In the framework of the present invention, it was unexpectedly found that certain agents (e.g. viruses, virus components or other compounds) that exhibit the ability of certain viruses to enter eukaryotic cells provide a significant increase in the expression level of nucleic acid introduced into the cell as part of the complex. The solution according to the invention is unexpected due to the fact that the range of nucleic acid complexes capable of penetrating into the cell is very wide.

 Thus, this invention relates to a transfection composition for higher eukaryotic cells, comprising a nucleic acid complex and a substance with an affinity for nucleic acid, while it additionally contains an endosomolytic agent in an effective amount.

 An endosomolytic agent is a substance capable of being absorbed by a cell and transferred to the cytoplasm containing nucleoli in which they are located after entering the cell. This ability of the endosomolytic agent is hereinafter referred to as “absorption function”. This absorption function includes the ability to actively turn on the cell, that is, by using the receptor-dependent mechanism of endocytosis, or passively turn on by the cell, that is, by using the liquid phase or as a component of the nucleic acid complex, and the ability to destroy nucleoli, which is usually referred to as nucleolus endosomolysis.

 According to one embodiment of the invention, a virus or a virus component is used as an endosomolytic agent. Further, a virus or component of a virus is referred to as a “free" virus (component).

In the framework of the present invention, we studied the effect of increasing doses of free adenovirus on the degree of transmission of a constant amount of conjugate-transferrin and polylysine in HeLa cells using luciferase as a reporter gene. Increasing the degree of gene transfer by adding free adenovirus reached a maximum of 1 • 10 ⁴ virus particles per cell, that is, a number that corresponds to the approximate number of adenovirus receptors per HeLa cell. The increased expression of luciferase, which is up to 2000 times compared with the expression achieved by a single conjugate of transferrin and polylysine, is due to a larger dose of the virus. Another series of experiments was aimed at identifying the effect of limiting the amount of conjugate and DNA complexes in the presence of a constant dose of free adenovirus. It was found that uptake of adenoviruses by the cell led to an increase in the degree of gene transfer with transferrin / polylysine over a wide range of DNA amounts. The maximum intensity of gene expression achieved with the conjugate-DNA complex corresponded to the intensity achieved with 100 times less DNA in the case of using adenovirus to increase transfection efficiency.

 The effect of endovirus infection on gene transfer was studied using both uncomplexed DNA and complexes of DNA and polylysine or conjugate of transferrin and polylysine. In these experiments, it was found that adenovirus infection did not lead to a significant increase in the transmission of uncomplexed DNA during transfection. In contrast, DNA transfer as a complex with polylysine or as a complex with transferrin and polylysine conjugate significantly improved adenovirus infection. The maximum effect is achieved in the case of the use of the conjugate of transferrin and polylysine. Since the polycationic part of the conjugate not only serves to bind transferrin to DNA, but also causes significant structural changes within the DNA, these experiments did not first establish whether the detected action was the result of improved fluid transport of condensed polycation DNA or an improved delivery virus of the conjugate complex bound to the receptor and DNA. Therefore, experiments were conducted on the binding and determination of the sequence in order to identify the cause of the detected action. The binding of the complex of conjugate of transferrin and polylysine and DNA or the complex of polylysine and DNA at low temperature allowed the removal of the excess complex in the liquid phase before infection with adenovirus. When it was done in this way, the yield of the receptor-bound complexes of the transferrin conjugate and polylysine and DNA increased significantly as a result of the addition of adenovirus. In the case of the use of a complex of polylysine and DNA, this effect was not observed. Thus, what improves significantly is DNA entry through the receptor-promoting mechanism of endocytosis.

 In addition, an analysis of the specific function of adenovirus, which causes improved gene transfer by the receptor, was performed. Weak heat treatment of virions does not change their ability to bind to the corresponding cell membranes, but affects their ability to destroy the nucleolus after absorption by the cell.

 Thus, various effects on the binding of the virus and the entry of the virus into the cell could be detected in separate experiments. It was also found that the thermal inactivation of adenoviruses led to the complete destruction of their ability to improve gene transfer using the receptor as an intermediary. This suggests that the destruction of the nucleoli caused by adenoviruses is part of their entry into the cell, which specifically determines the improvement in the return of the gene using a conjugate of transferrin or polylysine. The fact that the strain of a virus defective in replication could provide improved gene expression confirms the assumption that this phenomenon is due not to the replication function, but to the absorption function of the virion.

 To find out that the improvement of gene expression can be attributed to the possible transactivation of the introduced gene by the virus, experiments were carried out using a cell line, which in its structure is capable of expressing the RSV-LTR luciferase gene. In this cell line, adenoviruses show no effect, while in the parent cell line in which the gene was introduced using the conjugate of transferrin and polylysine, a significant increase in gene expression was observed. The results of these experiments show that adenovirus affects the event that takes place before transcription, and that its effect on improving gene transfer is not manifested at the level of gene expression, but at the level of gene transfer.

 In the framework of this invention, studies have also been conducted aimed at identifying the effect of free adenovirus on gene transfer using conjugates of transferrin and polylysine in selected cell lines. It was found that the presence of transferrin receptors on the target cell is necessary, but not sufficient in each case in order to ensure gene transfer using the conjugate of transferrin and polylysine. It is suggested that cell-specific factors related to the result of conjugate and DNA complexes in the nucleolus are an extremely important determining factor for the level of gene transfer achieved in this way. In this regard, experiments were carried out on selected cell lines aimed at studying both gene transfer using transferrin and polylysine conjugates and improving gene transfer using adenoviruses. Cells of the mucofiscidosis cell line (CFT1) showed a small level of luciferase gene expression after treatment with DNA and conjugate complexes of transferrin and polylysine. This expression level increased significantly as a result of treatment with d1312 adnovirus. In contrast, KB cells treated with DNA and conjugate complexes of transferrin and polylysine showed an expression level of the luciferase gene only slightly above the background level, despite the presence of the transferrin receptor. However, as a result of treatment with adenovirus d1312, luciferase activity in these cells was immediately detected. Adenovirus treatment of HeLa cells had a similar effect, although this effect was significantly stronger in these cells. Since HeLa and KB cells have approximately the same number of adenovirus receptors, the difference in improved gene transfer may be due to the number of transferrin receptors specific to each cell type. In contrast to these results, cell lines WI-38 and MRC5), which are known to only weakly support adenovirus infection, showed in the presence of d1312 only a slight improvement in gene expression after treatment with a complex of DNA and conjugate. Therefore, treatment with a free virus, for example, adenovirus, obviously leads to improved gene transfer using DNA and conjugate complexes in such cases where gene transfer is possible due to endocytosis carried out in the presence of a receptor, as, for example, in the case of CFT1 cells, and also in some cases where gene transfer this way is ineffective, as, for example, in the case of HeLa and KB cells. The level of improvement achieved varies significantly among different target cells, so it can be assumed that this effect is a function of both the number of virus receptors, for example, adenovirus receptors, in a particular cell, and the number of transferrin receptors.

 In the case of the use of a free virus, a substance with an affinity for nucleic acid, preferably an organic polycation, is preferably associated with a factor that promotes cell uptake. However, within the framework of the present invention, it was also found that DNA in the form of a complex with one substance with an affinity for nucleic acid, that is, without a factor promoting the uptake of the cell, under certain conditions can be introduced into the cell in the presence of a free virus. In addition, it was found that in the case of some cell lines, complexes consisting of nucleic acid and a substance with an affinity for nucleic acid can be introduced through the liquid phase under the condition of a sufficiently high concentration of the complex. The experiments that were carried out in the framework of this invention showed that an essential feature for the absorption capacity of nucleic acid complexes is their compactness, which can be attributed to the condensation of a nucleic acid by a substance with an affinity for nucleic acid. If a substance with a nucleic acid affinity has sufficient ability to bind to the cell surface in order to penetrate the cell with the virus, as well as to communicate a complex of mainly diseased electroneutrality and nucleic acid condensation to a compact structure, then there may be no need to improve the ability to penetration into the cell due to covalent binding of the factor that promotes the absorption of the cell with a substance with an affinity for nucleic acid in order to ensure the transfer of the complex into the cell by endocytosis carried out in the presence of a receptor as an intermediary. Many cells exhibit a relatively large affinity for certain substances with an affinity for nucleic acid, so that nucleic acid conjugates and binding factors are transferred to the cell without the need for a cell uptake promoting factor. This is true, for example, for hepatocytes that absorb complexes of DNA and polylysine.

 According to a preferred embodiment of the invention, a virus associated with a substance with an affinity for nucleic acid and having the ability to enter the cell as part of the conjugate-nucleic acid complex and to transfer the contents of the nucleoli into the cytoplasm in which the complex is located after entry into the cell is used as an endosomolytic agent .

 According to another preferred embodiment of the invention, a virus component is used as an endosomolytic agent associated with a substance with an affinity for nucleic acid and having the ability to enter the cell as part of the conjugate-nucleic acid complex and to transfer the contents of the nucleoli in which the complex is located after entry into the cytoplasm into the cage. Viruses and virus components that are linked to a nucleic acid binding domain are hereinafter referred to as "viral conjugates" (despite the type of binding).

 Viral conjugates, which are also the subject of this invention, contain the virus or virus components as an integral part of the functional construct. They combine the advantages of vector systems based on conjugates that promote cell uptake of the factor with the benefits that viruses contribute to these systems.

 In addition, the viral conjugates according to the invention have the advantage that, in contrast to the known conjugates that can be used for gene transfer using endocytosis in the presence of receptors as a mediator, they have a specific mechanism for their isolation from the cell vesicle system.

 The vector system using viral conjugates represents a qualitatively new approach compared to recombinant viral vectors, which consists in the fact that the transported foreign DNA is located on the outside of the virion. Therefore, the viral conjugates according to the invention can transport very large gene constructs into a cell without any limitation in the sequence.

 The suitability of a virus, which can be used as a free or bound virus or part of a virus as a viral component in the framework of the present invention, is determined by the aforementioned absorption function. Suitable viruses are, for example, those viruses that are capable of entering the cell by endocytosis in the presence of a receptor as an intermediary during transfection of cells with a nucleic acid complex and thereby isolating and thereby isolating the nucleic acid from the nucleolus into the cytoplasm. Such viruses are those viruses that are mentioned in the framework of this application, as well as other viruses capable of being absorbed by a certain cell, and those viruses that can carry out the isolation of the contents of the nucleolus in the cytoplasm.

 The susceptibility of the cell line to virus transformation as a means of facilitating the entry of the conjugate as a "free virus" depends on the presence and number of receptors on the surface of the target cell.

 Suitable viruses include those that are capable of penetrating into the cell via endocytosis in the presence of a receptor as an intermediary during transfection of cells with a nucleic acid complex and to carry out their isolation and thereby isolate the nucleic acid from the nucleolus into the cytoplasm. Without claims in part of this theory, we can say that in the case of the use of a free virus, this endosomolysis activity could have a beneficial effect on the nucleic acid complex transferred to the cell, which consists in the fact that these complexes are transported together with viruses from the nucleolus to the cytoplasm, that they enter these nucleoli, like viruses. If the complexes contain the virus in a bound form, then they benefit from the endosomolytic activity of the virus and are transported from the nucleoli to the cytoplasm. This results in fusion between the nucleoli and lysosomes and thus the enzymatic degradation that usually occurs in these cell organelles.

 In particular, viruses that are suitable for carrying out the invention and whose absorption function at the beginning of the infection is carried out by endocytosis in the presence of a receptor as an intermediary are lipid-free viruses, such as, for example, adenovirus, poliovirus, rhinovirus, as well as enveloped viruses such as, for example, vesicular stomatitis virus, Semliki Forest virus, influenza virus. Suitable are also pH-dependent strains of Moloney virus. In particular, viruses from the group consisting of adenovirus, subgroup C, type 5, Semliki Forest virus, vesicular stomatitis virus, poliovirus, rhinoviruses and Moloney leukemia virus are preferred.

 The use of RNA viruses that do not have reverse transcriptase within the scope of the present invention has the advantage that transfection in the presence of such a virus does not lead to the formation of viral DNA in the transfected cell. As a result of experiments according to the invention, it was found that the rhinovirus HRV2, a representative of the picornavirus groups, exhibits increased expression of the reporter gene. The effectiveness of rhinovirus was manifested both in free form and in the form of conjugates.

 For the purposes of this invention, the term “virus” (provided that it is absorbed by the cell and releases the contents of the nucleoli into which it enters) is understood in addition to wild types of mutants that, as a result of one or more mutations, have lost certain wild-type functions (other than absorption function), in particular the ability to replicate.

 Mutants are formed in the processes of ordinary mutagenesis due to mutations in protein regions that are responsible for replication functions and which can be supplemented by a packaging line. In the case of adenovirus, such mutants are ts mutants (thermosensitive mutants), E1A and E1B mutants, mutants that show mutations in MLP-stimulated genes, and mutants that show mutations in regions of certain capsid proteins. Viral strains that have corresponding natural mutations are also suitable. The ability of viruses to replicate can be investigated by methods known from the literature. So, for example, suspensions with various virus concentrations are fed to cell cultures and the number of dissolved cells, which can be seen as spots, is recorded.

 Other viruses that can be used in the framework of this invention are so-called defective viruses, that is, viruses that do not have the function required for autonomous replication of the virus in one or more genes for which they require the presence of a helper virus. Examples of this type of virus are defective interfering particles, which are derivatives of the infectious standard virus, have the same structural proteins as the standard virus, have mutations and need the standard virus as a helper virus for replication. Satellite viruses are also examples of such a virus group. Another group is the class of parvoviruses, which are designated as adeno-associated viruses.

 Since the cycles of penetration into the cells of many viruses are not fully characterized, there are apparently other viruses that will exhibit the endosomolytic activity required for their suitability in the framework of this invention. Live vaccines or vaccine strains are also suitable within the scope of this invention.

 The term "virus" is understood to mean inactivated viruses, for example, viruses inactivated by chemical treatment, such as, for example, formaldehyde treatment, UV irradiation, chemical treatment in combination with UV irradiation, for example, psoralen treatment and UV irradiation, γ-ray irradiation, neutron bombardment. Inactivated viruses, for example, those also used for vaccines, can be obtained by methods known from the literature, after which they can be examined for the suitability of increasing the transfer of DNA complexes. In the framework of the present invention, experiments were carried out in which adenovirus preparations were inactivated using a standard UV sterilization lamp or by treatment with formaldehyde. It was unexpectedly found that the degree of inactivation of viruses greatly increases the degree of reduction in gene transfer, which is achieved by adding adenovirus to the transfection medium. In these experiments, drugs inactivated by treatment with psoralen and UV-irradiation of biotinated adenovirus associated with polylysine associated with streptavidin were used. These experiments showed that inactivation led to a decrease in the titer of the virus, which significantly exceeds the decrease in gene transfer capacity. This is a clear indication that the mechanisms associated with normal replication in an active virus can be destroyed without adversely affecting the effect needed for gene transfer.

 The term "virus components" refers to parts of viruses, for example, the protein part free of nucleic acid (an empty capsid of the virus that can be obtained by recombinant methods), proteins obtained by fractionation, or peptides that have endosomolytic functions of the intact virus. These components of the virus can be obtained synthetically, or by peptide synthesis, or by recombinant methods, depending on their size. As a result of experiments within the framework of the present invention, it was found that adenovirus proteins linked through biotin / streptavidin to polylysine allow improved gene transfer. An example of fragments or proteins other than viral adenovirus, which are essential for cell inclusion, is influenza hemagglutinin (HA). The N-terminal sequence of the HA2 hemagglutinin subunit of the influenza virus is responsible for the isolation of the virus from the nucleolus. It was found that peptides consisting of 20 amino acids of this sequence are capable of fusion of lipid membranes and their partial destruction. Authentic and modified peptides of the influenza virus have been successfully used in the framework of this invention. Another example are retroviral envelope proteins, for example, HIV gp41 or parts of such proteins.

 The use of viruses, which as such are capable of penetrating into cells and thus act as a contributing factor to cell uptake, is a further aspect of the present invention.

 Viruses or virus components that alone cannot bind to and penetrate the cell are preferably used as viral conjugates as defined above. As a result of binding to a DNA-binding domain, for example, a polycation, a virus or a component of the virus, a large affinity for the DNA molecules is reported, so that it forms a complex with them and is transferred to the cell as a component of the nucleic acid complex, which also contains a cell absorption promoting conjugate factor and DNA binding domain. In addition to the transfer effect achieved with this, the binding of a virus or component of a virus to a nucleic acid binding domain can also lead to an improvement in its endosomolytic properties.

 By selecting another factor that promotes cell uptake, almost any higher eukaryotic cell can be transfected with the proposed system.

 As a result of a simple screening, it can be determined whether a given virus or a virus component exhibits an absorbing function and whether it can be used to carry out the present invention. Such an analysis, for example, to study a virus for its suitability as a free virus, target cells are brought into contact with a DNA complex in the presence or absence of the virus. The amount of DNA complex that is carried into the cytoplasm can then be easily determined by detecting a marker gene product, for example, luciferase. If the presence of the virus leads to the fact that the DNA complex is absorbed and introduced into the cytoplasm at a higher level than without the virus, then this can be attributed to the absorption function of the virus. It is also possible to compare the absorption level of the DNA complex in the presence of the test virus with another virus of which a suitable absorption function is known, for example, with subgroup C, type 5, adenovirus. Viral conjugates may also be subjected to such experiments. In these experiments, you can also explore additional parameters, such as, for example, conjugates with various factors promoting the absorption of cells in different quantities. In addition, a specialist in this field can easily carry out this type of experiment, if necessary in combination with other experiments, for example, experiments to determine liposome leakage, in order to study the components of the virus and other components with potential endosomolytic activity on their ability to improve gene expression.

 If intact viruses are used, then experiments are conducted aimed at identifying the ability of the virus to replicate. These experiments are preferably carried out in parallel with preliminary experiments aimed at identifying the ability of the virus to improve gene transfer. A study of the ability to replicate is carried out by known methods, such as, for example, the method of staining, the method for determining cytopathogenic effects or the method for determining late gene expression in the case of cytopathogenic viruses or viruses, which greatly contribute to the growth of host cells. For other viruses, methods specific to the virus are used, for example, a method for determining hemagglutination, or chemical-physical methods, for example using an electron microscope.

 In the framework of the present invention, viruses that can be obtained with a high titer, which are resistant, have a low pathogenicity in their native state and, if necessary, allow the elimination of replication ability, are used as preferred viruses, in particular those that are used as free viruses. Adenoviruses are particularly preferred. If a specific cell population is subject to transfection, then viruses that exhibit a specific ability for this population can be used. If different types of cells are transfected, then viruses that are infectious to a wide range of cells can be used.

 The requirements for free virus compositions are essentially that the virus should be of the highest purity and that a stabilizing buffer should be used that is selected based on the virus.

 In any case, according to the invention, only those viruses or virus components can be used in which the safety risk is as low as possible, in particular the risk of virus replication in the target cell and recombination of viral DNA with the host DNA.

 It is preferable to use the mechanism of penetration into the cells of viruses infecting animals differently than humans to improve the absorption and transfer of DNA into higher eukaryotic cells, in particular humans, while the virus is active in the destruction of the nucleolus in higher eukaryotic cells. Members of the adenovirus family were isolated from birds, amphibians, and several other animals. Adenoviruses from birds, amphibians, cattle, dogs, mice, sheep, pigs and monkeys, as well as human adenoviruses, can be obtained from the American Type Culture Collection in Rockville, Maryland.

 One possible advantage of using a virus, such as an adenovirus, from a rare species can be reduced toxicity in the target cells (for example, reproduction or early initiation of gene expression in mammalian cells cannot be expected from chicken or frog adenovirus), a reduced risk to the person involved in obtaining the rare adenovirus compared to human adenovirus, and reduced interference in the target organism from antibodies against human adenovirus or mice. The absence of interference with human or mouse antibodies is especially important when using viruses in gene therapy in humans and mice.

 Large adenovirus CELO does not show reactivity against antibodies that recognize the main group of epitopes of adenoviruses that infect mammalian cells. In addition, CELO adenovirus can be grown in fertilized eggs to produce a high yield (0.5 mg / egg). In the experimental part, it is shown that the conjugates of CELO and polylysine improve the transfer of DNA into HeLa cells, at a level that can be compared with the level achieved with the use of human adenovirus d1312. Thus, the use of CELO conjugates to improve DNA transport can be seen as a promising approach in experiments on gene therapy in humans. Rare species viruses are preferably used as components of viral conjugates in the combined complexes of the invention.

 The binding of the virus to the nucleic acid binding domain in the virus-containing conjugates of the invention may be covalent or non-covalent, for example, using a biotin-streptavidin bridge. It is also possible ionic binding in the case of the virus with acidic sites in surface proteins, which allows the connection with polycation.

 In the framework of this invention, complexes were formed under conditions that allow ionic interaction between adenovirus and polylysine before complexing with DNA. At the same time, control experiments were carried out under conditions in which polylysine was first neutralized by DNA and therefore it could not bind adenovirus. Adenovirus ion-binding complexes showed better results.

 Examples of viral components in the endosomolytic conjugates of the invention are empty viral capsids and viral peptides. The binding of the viral component to the nucleic acid binding domain may be covalent, for example, by chemically binding the viral peptide to polylysine, or non-covalent, for example, ionic in the case of the viral component, to acid residues that bind to the polycation.

 The ratio of a virus or a component of a virus to a substance with an affinity for a nucleic acid can vary widely. In the case of a peptide conjugate of influenza hemagglutinin and polylysine, gene transfer can be improved in a larger size with a high content of viral peptide in the conjugates.

 Conjugates of a virus or viral component and a nucleic acid affinity substance can be obtained (as well as conjugates of a factor that promotes cell uptake and polycation) by binding the components or by a recombinant method if the viral components and the nucleic acid binding domain are polypeptides.

 The binding of a virus or viral proteins or peptides to polyamines by chemical methods can be carried out by a method known for binding peptides, and if necessary, individual components are provided with binding agents before the binding reaction (this measure is necessary when there is no functional group suitable for binding, for example, a mercapto group or alcohol group). Binders are bifunctional compounds that first react with the functional groups of the individual components, and then bind the modified individual components.

Linking can be done using
a) disulfide bridges, which under reducing conditions can again split (for example, when succinimidyl pyridyldithiopropionate is used);
b) compounds which are generally stable under biological conditions (for example, thioethers, by the reaction of maleimido-binding substances with sulfhydryl groups bound to the second component of the binding substance);
c) bridges unstable under biological conditions, for example, ester bonds, or acetal or ketal bonds unstable under weakly acid conditions.

 In the framework of this invention, the endosomolytic peptides of influenza hemagglutinin HA2 are chemically coupled to polylysine using succinimidyl pyridyldithiopropionate. It was found that modification of the peptide with polylysine leads to an increase in endosomolytic activity. Transfection experiments showed that the efficiency of gene transfer in the presence of a conjugate of transferrin and polylysine as a mediator is significantly increased if the DNA complex, in addition to transferrin / polylysine, also contains conjugates of the peptide of influenza virus and polylysine.

Within the scope of this invention, adenovirus has bound polylysine in a variety of ways. One method for the formation of the conjugate of the virus and polylysine was carried out similarly to the formation of conjugates of transferrin and polylysine (see Wagner et al., Proc. Natl. Acad. Sci., USA 87, 1990, pp. 3410 - 3414) after modification of the defective adenovirus d1312 using heterobifunctional reagent. Unbound polylysine was removed by centrifugation. The binding ability of DNA was studied using radioactively labeled DNA (using K562 cells, it was found that in the absence of chlorocrin, significantly greater gene transfer is achieved in the presence of complexes consisting of DNA, adenovirus bound to polylysine, and transferrin bound to polylysine than in the presence of unmodified adenovirus, which is not associated with DNA.In addition, it was found that significant gene expression took place already in the presence of 0.0003 μg / DNA in 5 • 10 ⁵ HeLa cells using modifications polylysine adenovirus).

 If a virus or viral component (or an additional factor that promotes cell uptake, such as in the case of transferrin) contains suitable carbohydrate chains, they can be linked to a substance having an affinity for nucleic acid via one or more carbohydrate chains of a glycoprotein.

 Another preferred method for preparing the viral conjugates according to the invention is the enzymatic binding of a virus or viral component to a substance with an affinity for nucleic acid, in particular polyamine, using transglutaminase.

 The transglutaminase category includes a number of different enzymes that are found, for example, in the epidermis (epidermal transglutaminase), blood (factor XIII), and in cells of various tissues (tissue transglutaminase, for example, liver transglutaminase).

 Transglutaminases catalyze the formation of ε- (γ-glutamyl) lysine bonds in the presence of calcium ions with the breakdown of ammonia. A prerequisite is the presence of glutamines and lysines in proteins capable of interacting with the enzyme. In addition to the ε-amino groups of lysine, (poly) amines, such as, for example, ethanolamine, putrescine, spermine and spermidine, can also be used as a substrate.

 It is still not clear what factors are responsible for the fact that glutamine or lysine protein or polyamine reacts with the enzyme. It is only known that polyamines can be associated with numerous cellular proteins, such as, for example, cytokeratins, tubulin, cell membrane proteins, surface proteins of influenza viruses using transglutaminase.

 In the framework of the present invention, it was found that polylysine can bind to adenoviruses using transglutaminase. In this case, the binding process can be carried out in the presence of glycerol, which has the advantage that the preparation of a virus, for example, adenovirus, can be fed directly to the binding, since the buffer contained in it includes glycerin. The use of conjugates of adenovirus and polylysine, available as a complex with plasmid DNA and conjugates of transferrin and polylysine, allows expression of a gene that is many times greater than the effect achieved with the use of conjugates of transferrin and polylysine in the presence of non-polylysine-bound adenovirus.

 Another preferred method for preparing the conjugates of the invention is to bind the virus or its component to the polycation via a biotin-protein bridge, preferably via a biotin-streptavidin bridge.

 The known strong association of biotin with streptavidin or avidin is used to bind adinovirus with polylysine by modifying the adenovirus with biotin and chemically bind streptavidin with polylysine.

 The complexes consisting of DNA and a conjugate of streptavidin and polylysine, to which the biotin-modified virus is bound, and, if necessary, not covalently bound polylysine, exhibit very high transfection efficiency, even at low DNA concentrations. Particularly effective complexes are formed due to the fact that the virus modified with biotin is first bound to the conjugate of streptavidin and polylysine, after which DNA is bound. Binding to biotin can also be carried out using avidin.

 The relationship between the virus or its component and polylysine can also be carried out as follows. The virus is biotinylated, anti-biotin antibodies bind to polylysine and the link between biotin and antibody is used to bind the virus and polylysine. In this case, commonly used standard polyclonal or monoclonal antibodies against biotin are used.

 The binding of the virus to polylysine can also be accomplished by binding of the polylysine to a lectin having an affinity for the surface glycoprotein of the virus, the binding process in such a conjugate being carried out using the link between the lectin and the glycoprotein. If the virus does not have suitable side carbohydrate chains, then it can be modified accordingly.

 The virus can also be associated with a substance with an affinity for nucleic acid due to the fact that the surface of the virus is modified with an antigen foreign to the virus (for example, digoxygenin, a product of the Beringer Mannheim foreign company, DE, or biotin) and the connection between the modified virus and the substance with an affinity for nucleic acid set through an antibody that binds to the antigen. The particular implementation of the conjugate formation process of the invention depends on various criteria. For example, binding using biotin is the least specific and therefore widely used method, while binding using biotin leads to the formation of a very strong non-covalent bond. The enzymatic reaction with transglutaminase has the advantage that it can also be carried out on a very small scale. Chemical bonding is usually resorted to when large quantities of conjugate are to be synthesized. This method is usually the best for binding proteins or peptides of the virus. If inactivated viruses are used, inactivation is usually carried out before binding, provided that inactivation does not adversely affect binding.

 If a virus, for example, an adenovirus, or its component with endosomolytic activity, has available binding domains, for example, acidic domains for binding to a polycation, then the binding of the virus or its component to a polycation can also be carried out by ion. In this case, the positive charges of the polycation, which can be in the form of a conjugate with a factor that promotes cell uptake, are partially neutralized by the acid domain of the virus or its component, and the remaining positive charges are neutralized by nucleic acid.

 If the substance with an affinity for the nucleic acid is an intercalation substance, then it is modified with a binder suitable for the binding of a particular virus or its component. So, for example, in the case of binding by transglutaminase, it is modified with a spermine or a bifunctional group suitable for chemical bonding, for example, with an active ester group.

 The ratio of the virus or its component and nucleic acid binding substances can vary widely. It is usually determined empirically, for example, by binding a certain amount of the virus or its component with a different amount of polylysine. In this case, the optimal conjugate is used for transfection.

 According to another embodiment of the invention, a virus component, for example a viral peptide with endosomolytic activity, can be modified so that it can be directly linked to DNA. In this case, the peptide itself may contain a DNA binding domain obtained by synthesis of the peptide and incorporation into it, preferably by extension, of positively infected amino acids, preferably at the C-terminus.

 According to another embodiment of the invention, the endosomolytic agent is a non-viral, optionally synthetic peptide. If the proposed system contains a peptide of this type, then it is preferably ionically coupled to a substance with an affinity for nucleic acid, for example, polylysine in the presence of DNA complexes that promote the absorption of factor and polylysine by the cell. Moreover, the incorporation of the endosomolytic peptide into the nucleic acid complex is carried out by binding to a positively charged nucleic acid binding domain, preferably polylysine, through its acidic amino acid residues.

 Depending on the chemical structure of the peptide, in particular its end group, binding to polylysine can also be carried out by methods used in the framework of the present invention for the binding of peptides to polylysine. If a natural peptide is used, it can be modified with a suitable amino end group to make it suitable for binding.

 Another method for incorporating non-viral endosomolytic peptides into nucleic acid complexes is to provide them with sequences capable of binding to DNA. Such a sequence should be in the place where it does not adversely affect the endosomolytic activity of the peptide. Therefore, for example, peptides whose N-terminus is responsible for this activity are extended by DNA binding sequences at the C-terminus. Such sequences may be homologous or heterogeneous cationic oligopeptides, for example, an oligolysin tail, or a natural DNA binding domain, for example, a peptide derived from histone. These DNA binding sequences as an integral part of the endosomolytic peptide preferably contain about 10 to 40 amino acids. This embodiment of the invention makes it possible to increase the ratio of the endosomolytic sequence to the DNA binding sequence as compared to conjugates of a peptide with a high polycation content in order to achieve greater efficiency of the complexes.

 Non-viral endosomolytic peptides must meet the following requirements. With respect to endosomolytic activity, the leakage of lipid membranes achieved by the peptide should preferably be greater at low pH (5-6) than at pH 7. In addition, the destroyed areas of the membrane should be large enough to allow passage of DNA complexes (small pores are insufficient). In order to determine whether a particular peptide meets these requirements, in vitro experiments can be carried out due to the fact that the peptides are used in free or bound form and / or in the form incorporated in the DNA complex. In such experiments using liposomes, red blood cells, or cell cultures, an increase in gene expression is determined. Such experiments are the subject of exemplary embodiments of the invention. The optimal amount of peptide can be determined in the framework of preliminary titrations by analyzing the effectiveness of the resulting gene transfer. It should be noted that the effectiveness of various peptides and the optimal composition of the complex depend on the type of cell.

 Membrane disruptive peptides typically contain amphipathic sequences, namely a hydrophobic region that can interact with the lipid membrane, and the hydrophilic region stabilizes the aqueous phase at the membrane disruption site.

 In nature, there are some examples of membrane-disrupting peptides, typically small peptides or peptide domains of large polypeptides. Such peptides can be classified according to their function in a natural context, namely either to membrane-destroying peptides (e.g., peptides of naked viruses) and / or to peptides causing membrane fusion (e.g., enveloped viruses). In the case of synthetic peptides, both classes of peptide sequences are suitable for the destruction of the nucleoli. Most natural peptides are capable of forming amphipathic α-helices.

 PH specificity can be obtained by incorporating acidic residues into the hydrophilic region of the putative amphipathic alpha helix such that the helix can only form at an acidic pH, but not at a neutral pH at which repulsion between negatively infected acidic residues prevents formation spirals. This property is also found in the case of naturally occurring sequences (for example, at the N-terminus of the HA-2 influenza virus).

 A fully synthetic amphipathic peptide with the ability to destroy the membrane depending on the pH value is known. This peptide (in free form) provides the formation of only small pores in the membranes, which allow only the release of compounds of small size. In the framework of the variant of the invention, according to which non-viral, if necessary synthetic, peptides are used, the following operations are usually carried out. The amphipathic peptide sequence is selected from the group of available natural or artificial peptides known to those skilled in the art. If necessary, acidic residues (Gin, Asp) are included in order to make the membrane-destructive activity of the peptide more specific with respect to pH (the double acid mutant of the influenza hemagglutinin peptide according to Example 35, designated p50). If necessary, acidic residues can also be included in order to improve the ability of the peptide to bind to polylysine. Such a polycation-binding domain can be obtained by including C-terminal acidic extensions, for example, oligo-Glu-tail.

 Endosomolytic peptides suitable for the practice of this invention can also be obtained by fusion of existing natural and artificial sequences. In the framework of this invention, experiments were carried out using various peptides that were produced from the synthetic GALA peptide. Some derivatives that were used in these experiments were obtained by combining the GALA peptide or its modifications with the flu peptide sequences or its modifications, for example, with the peptides designated EALA-Inf and EALA-P50 according to example 35.

 The length of the peptide sequence may be critical with respect to the stability of the amphipathic helix. Improving the stability of short domains, which are produced from natural proteins that lack stabilizing protein sites, can be achieved by lengthening the helix.

 The endosomolytic activity of peptides can be increased due to the formation of homodimers, heterodimers or oligomers. As a result of the experiments according to the invention, it was found that the P50 dimer exhibits much greater activity than the monomer.

 In addition, experiments also showed the effect of synthetic peptides on DNA uptake by conjugates of transferrin and polylysine. In this case, various peptides were synthesized, which were tested for the ability to leak liposomes and red blood cells, as well as their effect on the expression of luciferase in TIB 73 and NIH 3T3 cells.

 A non-peptide amphipathic substance can also be used as an endosomolytic agent. The requirements for the suitability of such a substance for use in the framework of this invention are basically the same as in the case of amphipathic peptides, namely the ability to be incorporated into a nucleic acid complex, pH specificity and so on.

 Another embodiment of the invention relates to complexes comprising a nucleic acid and a conjugate capable of complexing with a nucleic acid, which serve to transfer the nucleic acid to higher eukaryotic cells. The complexes are characterized in that the conjugate consists of a substance with an affinity for a nucleic acid and an endosomolytic agent associated with a substance with an affinity for a nucleic acid, wherein the endosomolytic agent is capable of being incorporated into the cell as part of the complex of the conjugate and nucleic acid and to release the contents of the nucleoli which the complex is located after entering the cell, in the cytoplasm.

 In the framework of the present invention, complexes are preferably used in which the nucleic acid is bound to a substance with an affinity for the nucleic acid so that the complexes are mainly electrically neutral. According to a preferred embodiment of the invention, a virus or a component covalently linked to a polycation is used as an endosomolytic agent.

 The endosomolytic conjugates used in the framework of this invention also encompass (in addition to conjugates in which endosomolytic agents are ionically coupled to a DNA binding domain) endosomolytic agents that directly bind to DNA, for example through their main extension, although this kind of “conjugate”, strictly speaking , is not obtained due to the coupling of the connection, that is, due to the binding of two compounds to each other. The function of this type of endosomolytic agent as a component of the proposed system is independent of whether they are synthesized by conjugation of the endosomolytic agent and the DNA binding domain or whether the DNA binding domain is initially contained in it.

 According to another preferred embodiment of the invention, in addition to the endosomolytic conjugate, the complexes contain another conjugate in which the substance with an affinity for the nucleic acid, in the case of the endosomolytic conjugate of the polycation, is usually the same polycation as in the conjugate, and it is coupled with a cell-absorbing factor exhibiting affinity for the target cell. This embodiment of the invention is used, in particular, when the target cell does not or only has a few receptors for the virus used as part of the endosomolytic conjugate. This variant of the invention is also used when a virus component is used, for example, a naturally occurring, possibly modified peptide, non-viral, if necessary synthetic endosomolytic peptide or virus of an unusual species that does not have the ability to penetrate into cells that are to be transfected. The presence of an additional conjugate that promotes cell uptake of the factor and binding factor leads to an improvement in the characteristics of endosomolytic conjugates due to the fact that they form complexes with a nucleic acid together with the second conjugate and are transferred to the cell as part of the resulting complex, which is referred to as the “combination complex” in the following or "triple complex". It is assumed that combinational complexes are absorbed by cells either by binding to a surface receptor specific for an additional factor promoting the uptake of the cell, or, for example, when a virus or its component is used, by binding to a virus receptor, or by binding to both receptors, absorption occurs as a result of endocytosis caused by the receptor. When endosomolytic agents are released from the nucleoli, the DNA contained in the complexes is also secreted into the cytoplasm, while avoiding lysosomal degradation.

 In the experiments carried out in accordance with this invention, almost all HeLa cells were transfected with free adenovirus. In the case of hepatocytes, efficiency could be further improved by using triple DNA complexes in which reporter DNA is present as a complex with polylysine and transferrin conjugates and is associated with adenovirus. In this case, the presence of an endosomolytic virus and a complex of the ligand and receptor in the nucleolus is ensured. Moreover, in almost all cells of the indicated type, for example, BNL. -CL2 and HepG2, transfection occurs. In this case, the receptors available on the cell surface for both the virus and transferrin can capture triple complexes of DNA. However, it is also possible that triple DNA complexes can penetrate into the cell solely due to the action of a combination of the cell ligand and receptor. This possibility was investigated in experiments in which transferrin-containing triple DNA complexes penetrated into K562 cells mainly through the transferrin receptor rather than through the adenovirus receptor.

Surprisingly, it was found that triple complexes transferred DNA into the cell even in the case of very low DNA concentrations. So, for example, in the case of 30 pg DNA / 3 • 10 ⁵ cells, 1.8 • 10 ⁴ light units are obtained (resulting from the expression of a plasmid encoding luciferase). This concentration means 60 DNA molecules and 1 plaque-forming unit of the virus per cell. Therefore, this invention provides for the efficient transformation of higher eukaryotic cells with very small amounts of DNA.

 The presence of viruses, their components or non-viral endosomolytic agents in DNA complexes as components of endosomolytic conjugates has the following advantages.

 1. The broad applicability of gene transfer technology using nucleic acid complexes, since the endosomolytic agent itself, in particular in the case of a virus or its component, can be a factor that promotes absorption by the cell or can also be translated into a complex with DNA and other absorption-promoting cell factor, for example, transferrin, asialofetuin. In this way, it is possible to use the positive effect of viruses even in the case of cells that do not have a receptor for the virus.

 2. Improving the efficiency of gene transfer, since the binding of endosomolytic conjugates to DNA ensures that they penetrate into the cells together. The coordinated process of absorption and release of viruses and DNA also reduces the amount of DNA and viruses necessary for efficient gene transfer, which is extremely important when using the proposed system in a living organism.

 The term "promoting cell uptake factor" as used herein refers to ligands or fragments thereof which, after binding to a cell, penetrate into it as a result of endocytosis, preferably caused by a receptor, or factors which bind and penetrate by fusion with elements of the cell membrane.

 Suitable contributing factors to cell uptake are transferrin ligands, conalbumin, asialoglycoproteins (such as, for example, asialotransferin, asialorozomucoid and asialofetuin), lectins, substances that contain galactose and penetrate into the cells using the asialoglycoprotein receptor, lysylinoprotein, modified LDL, lipoproteins that penetrate cells using receptors (apo B100 / LDL), viral proteins, such as, for example, the AIDS gp120 protein, antibodies or fragments thereof cell surface antigens, such as anti-CD4, anti-CD7, cytokines, such as, for example, interleukin-1, interleukin-2, tumor necrosis factor, interferon, colony-stimulating factor, growth factors, such as, for example insulin, epidermal growth factor, platelet-derived growth factor, transforming growth factor β, nerve growth factor, insulin-like growth factor I, prolan B, prolan A, growth hormone, prolactin, glucagon, thyroid hormones, α-2-macroglobulin protease and shoulderless toxins. Other examples are immunoglobulins or fragments thereof as ligands for the Fc receptor or antibodies against immunoglobulins that bind to surface immunoglobulins. Ligands can be of natural or synthetic origin.

 The following sets out the basic requirements for the suitability of the above factors within the scope of this invention.

 a) They can be absorbed by the cell into which the nucleic acid should be transferred, and their ability to penetrate does not deteriorate or only slightly deteriorate if they are coupled to a binding factor.

 b) They are capable of transferring a nucleic acid into the cell by the “carry on back” method in any way that they choose.

 The following experimental part illustrates the wide possibilities of using a factor promoting cell uptake or an additional factor promoting cell uptake in the framework of this invention, as exemplified by transferrin and polylysine conjugates, asialofetuin or galactose complexes and transferrin and polylysine conjugates, wheat germ agglutinin conjugates, conjugate conjugate complexes gp120 protein specific for T cells, complexes of transferrin conjugates and poly Isigny and anti-Bd7, as an example, and DNA-polylysine complexes which do not contain factor promoting the absorption cell. In addition, the possibility of carrying out the invention when using viral conjugates is illustrated by the example of DNA complexes and a polylysine-conjugated virus or its component that do not contain a conjugate of an additional factor that promotes cell uptake and a binding factor.

 The possibility or necessity of using a factor that promotes absorption by a cell when using a free virus as an endosomolytic agent or an additional factor that promotes absorption by a cell when using a virus or a component of a virus or non-viral peptide that is part of an endosomolytic conjugate as an endosomolytic agent to provide or improve the absorption process nucleic acid complexes can be detected by appropriate test experiments. In these experiments, parallel transfections with nucleic acid complexes are carried out, in one case without (additional) promoting the absorption of the factor by the cell, for example, in the case of complexes of the nucleic acid and the viral conjugate, and in the other case nucleic acid complexes with another conjugate consisting of the additional promoting the absorption of the cell are used a factor for which target cells have an appropriate receptor, and substances with an affinity for nucleic acid.

 The use of a cell-promoting factor or an additional cell-promoting factor (in the case of a combination complex) is determined, in particular, by the target cell, for example, surface-specific antigens or receptors specific to the cell, which thus allow the desired transfer of the nucleic acid into the cell.

 Suitable nucleic acid affinities within the scope of this invention are, for example, homologous organic polycations, such as, for example, polylysine, polyarginine, polyornithine, heterogeneous polycations having at least two amino acids with different positive charges, and they may also have different chain lengths, as well as non-peptide synthetic polycations, such as, for example, polyethyleneimine. Other suitable nucleic acid affinities are natural polycationic DNA binding proteins, such as, for example, histones, protamines or their analogs or fragments, as well as spermine or spermidine.

The length of the polycation is not critical, provided that the complexes are mainly electrically neutral. Polylysine has a preferred chain length of about 20-1000 lysine monomers. However, for a given DNA length, there is no critical polycation length. So, for example, if DNA consists of 6,000 base pairs (hereinafter “bp”) and 12,000 negative charges, then the following amounts of polycation are used per mole of DNA:
60 mol of polylysine with a molar weight of 200
30 mol of polylysine with a molar weight of 400
120 mol of polylysine with a molar weight of 100, and so on.

 Any specialist can choose a polycation with a suitable length and in a suitable molar amount by carrying out standard experiments taking into account the foregoing.

 Other suitable nucleic acid affinities as a component of the conjugates are intercalating agents, such as, for example, ethidium dimers, acridine, or intercolating peptides containing tryptophan and / or tyrosine and / or phenylalanine.

 To make up the qualitative composition of nucleic acid complexes, the nucleic acid that is to be transferred into the cell is usually first determined. First of all, the nucleic acid is selected taking into account the biological action that should be manifested in the cell, and in the case of using the invention for gene therapy, taking into account the gene or region of the gene to be expressed, for example, to replace a defective gene, or taking into account the gene sequence which is subject to braking. The nucleic acids that must be transferred to the cell may be DNA or RNA, since there is no restriction on the nucleotide sequence.

 If tumor cells are used as a cancer vaccine, the DNA to be introduced into the cell preferably encodes a substance modulating the immune system, for example, a cytokine, such as interleukin (hereinafter: “IL”) - 2, IL-4, γ-interferon (hereinafter: “IF ") the α-factor of tumor necrosis (hereinafter:" BACKGROUND "). It is also possible to use combinations of cytokine-encoding DNA, for example, IL-2 and γ-IF. Another useful gene that can be introduced into tumor cells may be a multi-drug resistant gene (hereinafter: "mdr").

 It is also possible to introduce into the cell two or more different nucleic acid sequences, for example plasmid-containing cDNAs, encoding two different proteins with control of suitable regulatory sequences, or two different plasmid constructs that contain different cDNAs.

 Therapeutically active inhibitory nucleic acids that are transferred to cells in order to inhibit specific gene sequences can be gene constructs that serve to transcribe anti-mRNAs or ribozymes. In addition, it is also possible to introduce oligonucleotides into the cell, for example anti-m-oligonucleotides. Anti-m-oligonucleotides preferably include at least 15 nucleotides. Oligonucleotides may also be multimers. If ribozymes are to be included in the cell, they are preferably used as a component of the gene construct, including stabilizing gene elements, for example, elements of the t-RNA gene.

 In addition to nucleic acid molecules that inhibit genes, such as viral genes, genes exhibiting a different inhibitory pattern can also be used. Examples include genes encoding viral proteins that have so-called trans-dominant mutations.

 As a result of gene expression in the cell, proteins are obtained that dominate the corresponding protein of this type and, thus, protect cells that acquire “cellular immunity” by inhibiting viral replication.

 Suitable are trans-dominant mutations of viral proteins that are required for replication and expression, for example, Gag, Tat and Rev mutants. The latter showed inhibition of AIDS retrovirus replication. Another mechanism for achieving intracellular immunity is the expression of RNA molecules, including the binding site of a significant viral protein, for example, the so-called TAR bait.

 Examples of genes that can be used in somatic therapy and that can be transferred to cells as a component of gene constructs are factor VIII (hemophilia A), factor IX (hemophilia B), adenosine deaminase, α-1 antitrypsin (pulmonary emphysema), or a regulatory gene transmembrane conductivity of cystic fibrosis.

 The magnitude of nucleic acids can vary widely. So, for example, gene constructs of about 0.15 kilobase base pairs (hereinafter: “cpb”) (in the case of a t-RNA gene including a ribozyme) up to about 50 cbp or more can be successfully transferred to cells using the proposed system. Small nucleic acid molecules can be used as oligonucleotides. In this regard, it should be noted that the application of the proposed system is not limited either in relation to the sequence of genes or in relation to their size.

 After selecting the nucleic acid, a substance with an affinity for the nucleic acid, preferably an organic polycationic substance, is selected so as to ensure the formation of a nucleic acid complex, and the resulting complex should preferably be mainly electrically neutral. If the complexes include a conjugate of an additional factor that promotes cell uptake and an affinity for nucleic acid, the polycationic component of both conjugates is selected taking into account the issue of electroneutrality.

 It is known that optimal transfer of nucleic acid into a cell can be achieved when the ratio of conjugate to nucleic acid is selected so that complexes of the factor that promotes the absorption of the cell and the polycation / nucleic acid are mostly electrically neutral. It was found that the amount of nucleic acid absorbed by the cell does not decrease if a certain amount of the transferrin conjugate and polycation are replaced by a non-covalently bound polycation. On the contrary, in some cases, you can even significantly increase the absorption of DNA. It was found that DNA complexes exist in the form of toroidal structures with a diameter of 80-100 nm. Thus, the amount of polycation is selected taking into account electroneutrality and achieving a compact structure. In this case, the amount of polycation resulting from the implementation of nucleic acid charging, taking into account the provision of electroneutrality, usually also provide a compact structure of DNA.

 Thus, according to another embodiment of the invention, the complexes also include nucleic acid-binding substances in a non-covalently bound form, which may be identical or different from the binding factor. If the endosomolytic agent is a free virus, the complexes include a conjugate of nucleic acid and a factor promoting the uptake of the cell. In the case of the use of an endosomolytic conjugate, for example, a viral conjugate, the nucleic acid is converted into a complex with this conjugate, if necessary in combination with the conjugate, an additional factor that promotes cell uptake. The type and amount of non-covalently bound “free” substance with an affinity for nucleic acid is also determined by the conjugate or conjugates, in particular taking into account the binding factor in the conjugate. So, for example, if the binding factor is a substance that does not have or has only a limited ability to condense DNA, then in order to achieve a high degree of absorption of complexes by the cell, it is advisable to use substances with an affinity for DNA, which to a large extent possesses this property. But if the binding factor itself is a condensing nucleic acid substance and allows to obtain a compact nucleic acid structure sufficient to ensure efficient absorption by the cell, then it is advisable to use a substance with an affinity for nucleic acid, which provides enhanced expression due to other mechanisms.

 Suitable “free” nucleic acid affinity substances according to the invention are, for example, compounds capable of condensing the nucleic acid and / or protecting them from undesired cell degradation, in particular the aforementioned polycation type substances. Other suitable substances are those which, by binding to a nucleic acid, improve its availability to the cell expression system, thereby improving its transcription / expression. An example of such a substance is the chromosomal non-histone protein HMG1, which has the ability to communicate DNA of a compact structure and to facilitate expression in cells.

 When determining the molar ratio of an endosomolytic agent and / or a factor promoting the absorption of a cell and an affinity for a nucleic acid or nucleic acids by a cell, it should be taken into account that nucleic acid is complexed, the resulting complex can bind to the cell and be absorbed by it and that it is secreted from the nucleoli or using an endosomolytic agent or by itself.

 The ratio of the factor that promotes the absorption of the cell by the binding factor and nucleic acid depends, in particular, on the size of the polycation molecules and the distribution number of positively infected groups. Therefore, these criteria must be consistent with the size and structure of the transferred nucleic acid. The molar ratio of the factor promoting the uptake of the cell and the substance with a nucleic acid affinity is preferably about 10: 1 to 1: 10.

 After constructing and synthesizing the conjugates and determining the optimum for efficient transfection of the conjugate to DNA ratio, the amount of conjugate that can be replaced with a free substance with nucleic acid affinity can be determined by titration. If polycations are used both as a binding factor and as a free substance with an affinity for nucleic acid, then the polycations can be identical or different.

 Upon receipt of the proposed system, including conjugates of the virus, it is advisable to proceed as follows. First, a gene construct is selected that is to be transferred to the cell, and the virus or its component is suitable for this transfection. Then the virus or its component is bound to the polycation and transferred to the complex with the gene construct. Based on a certain amount of virus conjugate, it is possible to titrate by treating target cells with this (constant) amount of conjugate and reducing the DNA concentration, or vice versa. Thus, the optimal ratio of DNA to virus conjugate is determined. If an additional factor promoting the uptake of the cell is used, then the optimal ratio of the virus conjugate to the conjugate promoting the uptake of the factor can be determined by titration using a certain amount of DNA.

 Complexes can be obtained by mixing a nucleic acid, a virus conjugate and, if necessary, a conjugate that promotes cell uptake of the factor and binding factor, as well as a non-covalently bound material with an affinity for the nucleic acid. Moreover, all components can be in the form of dilute solutions. If polycations are used as a binding factor and simultaneously as “free” polycations, it is advisable to first obtain a mixture of conjugates with “free” polycations and then combine this mixture with DNA. The optimal ratio of DNA and conjugate or conjugates to polycations is determined by repeated titration, and a series of transfection processes is carried out using a constant amount of DNA and increasing amounts of a mixture of conjugate or conjugates and polycation. The optimal ratio of conjugate or conjugates to polycations in the mixture can be determined, for example, by comparing the optimal composition of the mixtures used in multiple titration processes.

 DNA complexes can be obtained at physiological concentrations of salts. Another possibility is the use of high salt concentrations (approximately 2M NaCl) and subsequent adjustment to physiological conditions by slow dilution or dialysis.

 The most appropriate sequence of mixing the components of a nucleic acid, conjugate or conjugates and, if necessary, a non-covalently bound material with an affinity for nucleic acid is previously determined in a series of relevant experiments. In some cases, it may be appropriate to pre-form a complex of nucleic acid and conjugate or conjugates, followed by the addition of a “free” substance with affinity for the nucleic acid, for example polycation, for example, in the case of conjugates of transferrin and dimeric ethidium and polylysine.

 According to a preferred embodiment of the present invention, transferrin is used as a cell-promoting factor and an additional cell-promoting factor, and a polycation is used as a binding factor. The term "transferin" refers to natural and synthetic transferrin, as well as modifications of transferrin, which bind to the receptor and are transferred to the cell.

 Nucleic acid is absorbed in the form of complexes in which the conjugates of the factor promoting the uptake of the cell and the polycations are in the form of a complex with the nucleic acid. If the complexes still include a non-covalently bound material with an affinity for nucleic acid, then this material is preferably a polycation. This second polycation is identical or different from the polycation contained in the conjugate or in both conjugates.

 In the case of "combination complexes", the nucleic acid is absorbed by the cell in the form of a complex in which conjugates of the factor promoting the uptake of the cell and endosomolytic conjugates are complexed with the nucleic acid. Conjugates of a factor that promotes cell uptake and polycation, which are used together with free virus or together with viral conjugates in combination complexes, can be obtained by the chemical method or by the recombinant method in the case of using the polypeptide as a polycation.

 In the framework of this invention, conjugates are preferably used in which a glycoprotein, for example, transferrin, and a binding factor linked to each other via at least one glycoprotein carbohydrate chain. In contrast to the conjugates obtained by traditional coupling methods, the conjugates obtained by this method are free from modifications derived from the binding agent used. In the case of the use of glycoproteins with at least one carbohydrate group suitable for binding, for example, transferrin, the resulting conjugates also have the advantage that their binding sites to glycoproteins and a binding factor are precisely defined.

 The amount of endosomolytic agent used and its concentration depend on the target transfection. It is advisable to use the minimum amount of virus or viral conjugate, which is necessary to ensure that the cell absorbs the virus or viral conjugate and the nucleic acid complex and release from the nucleoli. The amount of virus or viral conjugate should be consistent with this type of cell, and first of all, the effectiveness of the virus for this type of cell should be taken into account. Another criterion is the conjugate of the cell-promoting factor and binding factor conjugate, in particular with respect to the cell-promoting factor for which the target cell has a certain number of receptors. In addition, the amount of virus or viral conjugate depends on the amount of DNA transferred. Typically, a small amount of virus is sufficient to ensure stable transfection, which requires only a small amount of DNA. In contrast, in the case of transient transfection requiring more DNA, the virus should be used in larger quantities. In certain cases, it is advisable to conduct preliminary experiments with the use of target cells intended for transfection, if necessary in the form of a mixed population, and intended for transfection of the vector system in order to determine the optimal concentration of the virus by titration. In this case, it is advisable to use a gene construct as DNA, which in size mainly coincides with the substance intended for this particular case, and contains a reporter gene to more easily determine the efficiency of gene transfer. Suitable reporter genes for performing such experiments are luciferase and β-galactosidase.

 In the practice of this invention, the simultaneous use of a complex of nucleic acid and an endosomolytic agent is preferred. But it is quite possible and their consistent application. In the case of separate use, the sequence is not critical, provided that the activities are carried out one after another in compliance with only a short period of time to ensure effective simultaneous contact of the components.

 If the free virus is used as a separate preparation, then the simultaneous administration of the free virus preparation and the complexes can be ensured due to the fact that the virus preparations are part of the transfection medium containing the nucleic acid complex. In the case of simultaneous application of the free virus, the nucleic acid complexes and the virus preparation are pre-mixed with each other. According to a preferred embodiment of the invention, the endosomolytic agent is a component of the combination complex.

 In certain cases, it may be appropriate to use a lysosomatropic substance in addition to the endosomolytic agent, for example, if the endosomolytic agent is an endosomolytic conjugate of peptides or a retrovirus whose endosomolytic activity is independent of the acidic pH. It is known that lysosomotropic substances inhibit the activity of proteases and nucleases and therefore can inhibit the degradation of nucleic acids. Such substances are, for example, chloroquine, monensin, nigericin and methylamine. Monensin was found to increase expression of the reporter gene in the case of the use of Moloney retrovirus. The presence of chloroquine leads to the expression of a reporter gene introduced by DNA transfer in the presence of transferrin, almost 100% in K562 cells. BNL.CL2 cells or HepG2 hepatocytes do not respond to chloroquine; they can be transfected to a level of 5-10% if the endosomolytic properties of defective replication or chemically inactivated free adenovirus are used. This invention enhances the benefits of biological vectors. As a result of the distribution of receptors, tropism is obtained to promote the absorption of factor and virus by the cell. Coordination of these two components with the used cell population allows for higher selectivity, which, in particular, is important when applying the invention for therapeutic purposes.

 Another object of the present invention are pharmaceutical compositions that contain a therapeutically active nucleic acid complex as an active substance, preferably as parts of a gene construct, an endosomolytic agent (if necessary in the form of a conjugate) and, if necessary, a conjugate that promotes cell uptake of the factor. You can use any inert pharmaceutical acceptable carrier, such as, for example, saline, which may contain phosphate buffer, or any carrier in which the DNA complexes are soluble to the desired extent. A method of preparing a pharmaceutical composition is widely known.

 This invention provides maximum flexibility in its implementation, for example, when it is used in the form of a pharmaceutical composition. The proposed system may be in the form of a lyophilisate or contained in a suitable buffer in a deep frozen state. It can also be used as a ready-to-use reagent in solution, preferably in chilled form. The components necessary for transfection, i.e. DNA, an endosomolytic agent, if necessary in the form of a conjugate or ready to form a conjugate with an appropriate partner, a DNA-binding substance, if necessary, in the form of a conjugate with a factor promoting cell uptake, if necessary a free polycation, may be contained in a suitable buffer system or may be gradients of a transfection kit. If a transfection kit is used as a modular system, it may contain various DNAs, for example, DNAs encoding various antigens, and / or various conjugates of a factor that promotes cell uptake. The solution to the question of the use of components in the form of a ready-to-use preparation or their mixing immediately before use depends, among other things, on the stability of the complexes, which can be determined in advance by known methods. According to a preferred embodiment of the invention, the conjugate of adenovirus and polylysine obtained with transglutaminase, which is storage stable, can be used as an ingredient in the kit. According to another preferred embodiment of the invention, the biotinylated adenovirus and streptavidin / polylysine are mixed together before use.

 The proposed system can be applied systemically, preferably intravenously, as part of a pharmaceutical composition. The target organs to which this application is directed may be the liver, spleen, lungs, bone marrow, and tumors. An example of local application is pulmonary tissue (the use of the proposed system as parts of a pharmaceutical composition, which is a liquid for instillation or an aerosol for inhalation). In addition, pharmaceutical compositions can also be applied by direct injection into the liver, muscle tissue, tumor, or by local delivery to the gastrointestinal tract. Another method of administering the pharmaceutical composition is through the drainage system of bile. This method provides direct access to the membranes of hepatocytes on the tubules of bile, which avoids the interaction of the composition with blood components.

 Myoblasts (immature muscle cells) are known to be capable of transporting genes into the muscle fibers of mice. Since myoblasts can release a gene product into the blood, this approach can be more widely used than treating genetic defects in muscle cells, such as, for example, a defect expressed by muscular dystrophy. Consequently, suitably prepared myoblasts can be used to isolate gene products that either act in the blood or are transported by blood. The experiments in accordance with this invention showed that cultures of myoblasts and muscle tubes, even primary ones, can be subjected to highly efficient transfection. The most suitable transfection medium contained combination complexes of biotinylated adenovirus, transferrin / polylysine and streptavidin / polylysine. In addition to reporter luciferase and β-galactosidase genes, factor VIII was expressed in muscle cells. In addition, CELO chicken adenovirus was used in combination complexes containing wheat germ agglutinin as an additional factor promoting cell uptake.

 Therapeutic use can also be carried out ex vivo, and the treated cells, for example, bone marrow cells, hepatocytes or myoblasts, are returned to the body. Another ex vivo application of the proposed system is reduced to the application of so-called "cancer vaccines". The principle of this therapeutic approach is to isolate tumor cells from the patient and transfect cells with DNA encoding a cytokine. The next technique may be to inactivate the cells, for example, by irradiation, which is carried out in such a way that the cells no longer have the ability to replicate, but are still capable of expressing the cytokine. Genetically modified cells are then given as a vaccine to the patient from whom the cells were isolated. In the area of the vaccination site, secreted cytokines activate the immune system, for example, by activating cytotoxic T cells. These activated cells are capable of manifesting their action in other parts of the body and acting on untreated tumor cells. Thus, the risk of re-formation of the tumor and the formation of metastases is reduced.

 In the experiments of this invention, primary melanoma cells were successfully transfected with a reporter gene, which was contained in the combination complexes of polylysine-bound adenovirus and transferrin / polylysine.

 The present invention can also be used to determine the immunoreaction of a host to a given antigen. Such tests are based on gene transfer to antigen expressing cells. Antigen-specific cytotoxic T-lymphocytes (hereinafter: “CTLs”) that destroy infected cells play an important role in protecting the host from viral infections or tumors. The interaction between the T cell and the antigen representing cell (hereinafter “PAK”) is limited by human lymphocyte antigens (hereinafter: “LAC”), which are molecules of great tissue compatibility. In order to carry out a study of the degree of destruction of CTLs of antigen expressing cells in an in vitro experiment, it is necessary to present the CTL antigen in the correct context of LA. Such a study can be carried out as follows. PAAs are transfected with a DNA construct containing a sequence encoding an antigen. Antigen epitopes will bind to MHC class I and will be present on the cell surface as a target for a CTL-specific reaction. After incubation, along with a sample of the patient's serum, which depends on the presence of specific CTLs, PAA will be lysed. In this case, lysis is measured by determining the release of, for example, radioactive chromium, which is incorporated into the PAA before being added to serum.

 Induced B-lymphoplastoid cell lines can be used to express antigen genes by transfection with recombinant vaccine viruses. However, cells that efficiently express antigen within about one day die as a result of the lytic effect of the vaccine. These difficulties can be overcome by using the proposed system for introducing antigen-encoding DNA constructs, for example, constructs encoding AIDS antigens or tumor antigens, into fibroblasts in order to give them the ability to express antigen. Primary fibroblasts can be easily obtained by biopsy, easily grown and amenable to highly efficient transfection (with a degree of approximately 50 - 70%) of the proposed system. Such assays are useful for identifying vaccine-recognized epitopes by killer cells. In addition, they can advantageously be used to determine the limited LACH immune response of an individual to a given antigen.

 The invention can be used for the production of recombinant proteins, since in almost all cells a high degree of expression of the transferred genes is achieved. However, with respect to the sequences and molecular weight of the transferred DNA, there are only some restrictions, or there are no restrictions at all. There is a wide range of different kinds of cells that can be transfected with the proposed DNA constructs. For example, cells of almost any type can be used to produce recombinant proteins with high biological activity.

 Gene transfer to cells can be carried out in the same manner as shown for luciferase and α-interferon, that is, almost any gene construct that provides the desired protein product can be transported in this way. The desired protein product can be isolated from a transfected cell culture (either culture supernatant or a suitable homogenization product) 24 hours or one week after transfection. Isolation is also possible after a longer period after transfection.

 Using the proposed system for the production of recombinant proteins has the following advantages.

 1. Due to the high efficiency of transfection (more than 90% of transfected cells are capable of expressing the corresponding gene at a high level), preliminary selection of positively transfected cells is not required and, in addition, there is no need for the selection of stable cell lines. A small cell culture may be sufficient to produce the required amount of protein.

 2. Large gene constructs can be transported. Until now, constructs up to 48 kbp have been successfully transferred to cells.

 3. Gene expression can be performed in cells that provide suitable post-translational processing and modification (for example, vitamin A-dependent carboxylation of blood coagulation factors or glycosylation specific for the cell type).

 4. A wider selection of types of target cells for gene expression is possible.

 The invention is further described using the following examples, which are presented for purposes of illustration and not limitation of its scope. The following materials and methods are used in these examples unless otherwise indicated.

Obtaining complexes of DNA and conjugate of transferrin and polylysine
a) Conjugates of transferrin and human polylysine
A solution of 280 mg (3.5 μmol) of human iron-free transferrin in 6 ml of 30 mM sodium acetate buffer with a pH of 5 was cooled to 0 ^° C and 750 μl of 30 mM sodium acetate buffer with a pH of 5 containing 11 mg (51 μmol) of sodium periodate. The mixture was left to stand in the dark in an ice bath for 90 minutes. In order to remove low molecular weight products, gel filtration is carried out on Sephadex G-25 ionite. A solution is obtained containing approximately 250 mg of oxidized transferrin (determined using ninhydrin). (To prove the oxidized form containing aldehydes and giving a color reaction when stained with anisic aldehyde, the mixture is dropped into a thin layered silica gel plate, dried, the plates are immersed in a mixture of p-anisaldehyde, sulfuric acid and ethanol in a ratio of 1: 1: 18, dried and heated). The modified transferrin solution is quickly added (within 10-15 minutes) to a solution containing 1.5 μmol of fluorescein-labeled poly (L) lysine with an average length of 190 lysine monomers in 4.5 ml of 100 mM sodium acetate at pH 5. pH of the solution adjusted to 7.5 by adding 1 M sodium bicarbonate buffer. With an interval of one hour, 28.5 mg (450 μmol) of sodium cyanoborohydride are added 4 times to the mixture.

After 17 hours, add 2 ml of 5 M sodium chloride in order to bring the solution to a total concentration of 0.75 M. The reaction mixture is fed to a Mono S HR 10/10 cation exchange column from Pharmacy, Sweden, after which it is eluted with a salt gradient of 0.75 M-2.5 M sodium chloride having a constant content of 25 mm HEPES, pH 7.3. High salt concentration when fed to the column and at the beginning of the implementation of gradient elution is essential for obtaining conjugates. Some amounts of transferrin (approximately 30%) with weak fluorescence elute at the beginning of the process. The majority of the fluorescence-labeled conjugate was eluted at a salt concentration of 1.35-1.9 M and collected in 3 fractions. After two series of dialysis using 2 L of 25 mM HEPES buffer, pH 7.3, these fractions (in the order of their elution) give fraction A containing 45 mg (0.56 μmol) of transferrin modified with 366 nmol of polylysine (hereinafter referred to as conjugate "TfpL190A"), fraction B containing 72 mg (0.90 μmol) of transferrin modified with 557 nmol of polylysine (hereinafter referred to as "TfpL190B) and fraction C containing 7 mg (85 nmol) of transferrin modified with 225 nmol of polylysine (hereinafter this conjugate is designated "TfpL190C"). If the conjugates are not immediately served for further use ix, they were frozen in liquid nitrogen and stored at -20 ^o C in the iron-free form before the introduction of the iron sample. (0.5 - 1 mg) were adjusted to a physiological salt concentration (150 mM), addition of sodium chloride The inclusion of iron. carried out by adding 4 μl of 10 mm citric buffer of iron (III) (containing 200 mm citrate; the pH of the buffer was adjusted to 7.8 by adding sodium bicarbonate) per mg contained in the conjugate of transferrin. Iron-containing conjugates are divided into small aliquots before they are used to form a complex with DNA, instantly frozen in an atmosphere of liquid nitrogen or in a dry atmosphere of ice and ethanol and stored at a temperature of -20 ^o C (this approach justified itself, since it was found that repeating processes thawing and freezing lead to a loss of activity of conjugates).

b) Conjugates of mouse transferrin and polylysine
A similar method is used as in the case of human transferrin, while the binding is carried out using side carbohydrate chains. 4.1 mg (51 nmol) of mouse transferrin and 2.1 mg (34 nmol) of polylysine with a molecular weight of 290 (hereinafter: “pL 290”) are used. Get conjugates consisting of 15.5 nmol of mouse transferrin and 13 nmol pL 290.

Plasmid DNA
a) pRSVL DNA.

 Obtaining plasmid DNA pRSVL (containing the Photinus pyralis luciferase gene under the control of Rous Sarcoma LTR virus) is carried out using known methods using triton X. Next, equilibrium density gradient centrifugation using cesium chloride and ethidium bromide, decolorization with buganol-1 and dialysis using 10 mM Tris / HCl buffer (pH 7.5, 1 mM EDTA). The complex is formed by mixing 6 μg of plasmid DNA in 350 μl of HBS buffer (150 mM sodium chloride, 20 mm HEPES, pH 7.3) with 12 μg of conjugate of transferrin and polylysine in 150 μl of HBS buffer. Complexation is carried out 30 minutes before the complex is added to the cells.

b) pCMVL DNA
Plasmid pCMVL (a reporter gene construct containing the Photinus pyralis luciferase gene under the control of the cytomegalovirus promoter) is obtained by removing the BamHI insert from plasmid pSTCX556, treating the plasmid with the Klenov fragment, and introducing the treated HindIII / Ssp1 and Klen fragment from the plasmid pV plasmid . DNA is obtained analogously to the preparation of pRSVL DNA.

Getting the virus drug
a) Adenovirus preparations
Adenovirus strain d1312 having a deletion in the Ela zone is used. Virus replication is carried out in an Ela-trans complementing cell line 293 followed by purification in a known manner. The purified virus is fed to a storage buffer (100 mM Tris, pH 8.0, 100 mM sodium chloride, 0.1% bovine serum albumin (hereinafter: "ACC"), 50% glycerol) or to a HBS buffer containing 40% of glycerol and aliquots are stored at a temperature of -70 ^o C. The concentration of virions is determined by UV spectrophotometric analysis of the extracted genomic viral DNA (formula: one unit of optical density corresponds to 10 ¹² viral particles per ml).

b) The drug retrovirus
The Moloney N2 retrovirus of mouse leukemia is packaged in an ecotropic packaging line. The supernatants obtained from virus-expressing cells are collected, instantly frozen in an atmosphere of liquid nitrogen and stored at -20 ^° C. The supernatants used in the examples have a titer of about 10 ⁶ colony forming units per ml (hereinafter: “cfu / ml”), which was determined by the formation of neomycin-resistant colonies using NIH3T3 cells. The supernatants are passed through a FILTRON membrane located in a stirred cell concentration apparatus under nitrogen pressure. In this case, 10-30 ml of the supernatant is usually thickened 10 times.

Cells and environment
HeLa cells were cultured in DMEM supplemented with 5% inactivated heat treatment of calf fetal serum (hereinafter: “TES”), 100 units / ml penicillin, 100 μg per ml streptomycin and 2 mM glutamine. WI-38, MRC-5 and KB cells were cultured in EMEM (Igl-modified basic medium) supplemented with 10% inactivated heat treatment of TPP, antibiotics contained in DMEM, 10 mM amino acids and 2 mM glutamine. The epithelial cell line of respiratory cystic fibrosis CFT1 was cultured in F12-7X medium. To carry out gene transfer, cells are cultured in 6 cm diameter plates equipped with cells until a degree of growth of 5 × 10 ⁵ cells is achieved. The medium is removed and 1 ml of DMEM or EMEM / 2% TES is added. Conjugate and DNA complexes are then added, followed by immediate delivery of adenovirus d1312 (0.05 - 3.2 • 10 ⁴ particles per cell) or a comparable buffer volume (1 - 80 μl). The plates are returned to incubation at 37 ^° C. in the presence of 5% carbon dioxide for one hour, after which 3 ml of complete medium is added. After an additional 24-hour incubation, cells are harvested in order to determine the expression of the luciferase gene. In the case of CFT1 cells, cultivation in F12-7X medium is carried out for 4 hours without human transferrin before gene transfer.

 The cells obtained from the American ATCC collection have the following registration numbers: HeLa cells: CCL 2; K562 cells: CCL 243; HepG2 cells: HB 8065; TIB-73 cells: TIB 73 (BNL CL.2); NIH3T3 cells: CRL 1658; cells 293: CRL 1573; KB cells: CCL 17; WI-38 cells: CCL 75; MRC 5 cells: CCL 171. H9 cells with registration number 87 were obtained from the American organization AIDS Research and Reference Reagent Program, U.S. Department of Health and Human Services.

Primary lymphocytes are obtained by feeding 25 ml of a cord blood sample in an experimental tube containing EDTA. Under the aliquots, a 4.5 ml bottom layer of Ficoll-Hypac Pharmacia, Sweden was placed and centrifuged at 2500 rpm for 15 minutes. The brownish layer between the upper plasma layer and the transparent Ficoll layer is removed (approximately 10 ml). 40 ml of IMDM plus 10% TES are added, the sample is centrifuged at 1200 rpm for 15 minutes and the cell material is suspended in 50 ml of fresh IMDM plus 10% TES (cell density is about 2 x 10 ⁶ cells / ml). 250 μl aliquot of phytohemagglutinin is added, the culture is incubated at 37 ^° C for 48 hours in the presence of 5% carbon dioxide, and recombinant interleukin-2 (concentration: 20 units per ml) is added. Cells are diluted 1: 3 with IMDM plus 20% TES containing 2 units of interleukin-2 per ml. Aliquots of the cells are deep frozen in an atmosphere of liquid nitrogen in the presence of 5% methyl sulfoxide. Before use, cells are grown in IMDM medium containing 20% TES and 2 units of interleukin-2 per ml.

To carry out the subsequent binding process, HeLa cells are equilibrated at 4 ^° C in 1 ml of DMEM supplemented with 2% TES. Conjugate and DNA complexes were added in the same manner as in other cases, and plates were incubated at 4 ^° C. for 2 hours. Then the plates were washed intensively with ice cold DMEM containing 2% TES, after which 2 ml of this medium was added. Adenovirus d1312 or buffer is added, the cells are allowed to slowly heat up, after which they are fed for 24-hour incubation. After incubation, cells are collected and examined for luciferase gene expression.

Determination of luciferase activity
Preparation of cell extracts, standardization of protein content and determination of luciferase activity are carried out by widely known methods.

Example 1. Determination of the effect of adenovirus on gene transfer using conjugates of transferrin and polylysine. First, the effect of increasing doses of the virus on the ability of a certain amount of the conjugate and DNA complex to carry out gene transfer is examined. In order to form a complex, 6 μg of the plasmid pRSVL is mixed with 12 μg of the conjugate of human transferrin and polylysine (hereinafter: "hTfpL 190B"). The conjugate-DNA complex, as well as various amounts of adenovirus d1312 (0.05 - 3.2 • 10 ⁴ viral particles per cell) are added to HeLa cells. The results of this experiment are presented in FIG. 1. Luciferase activity is expressed in light units of 50 μg of total cellular protein. According to this analysis, increasing amounts of added adenovirus lead to a corresponding improvement in gene transfer. In FIG. presents average values of 2 to 4 individual experiments. Beams indicate standard deviation.

Example 2. The effect achieved depending on the amount of complex conjugate and DNA. DNA conjugate complexes in the form obtained in the same manner as in Example 1 of logarithmic diluted preparations are added to HeLa cells with or without a constant amount of d1312 adenovirus (1 • 10 ⁴ virus particles per cell). The luciferase activity is determined in the same manner as in Example 1. The experimental results are shown in FIG. 2.

 Example 3. The improvement in gene transfer achieved by the conjugate of transferrin and polylysine in the presence of adenovirus occurs through receptor-induced endocytosis.

a) The effect of adenovirus on the transfer of DNA available as a complex
The following components are used for transfection. 6 μg pRSVL DNA without transferrin and polylysine conjugate (DNA); 6 μg of pRSVL DNA and 6 μg of non-conjugated polylysine with a molecular weight of 270 (DNA + pL); 6 μg pRSVL DNA and 12 μg conjugate of transferrin and polylysine with a molecular weight of 190 used in the previous examples (DNA + hTfpL190B). These materials are added to HeLa cells together with adenovirus d1312 at a concentration of 1 • 10 ⁴ viral particles per cell (d1312) or without it. Obtaining cell extracts, standardizing the total cellular protein and determining the luciferase activity are carried out in the above manner. The results of the experiment are presented in FIG. 3a.

b) The effect of adenovirus on the transfer of DNA bound to the receptor
Conjugate and DNA complexes (DNA + hTfpll90B) or polylysine and DNA (DNA + pL) complexes are bound to HeLa cells by incubation at 4 ^° C. The unbound complex is removed before the addition of adenovirus d1312 at a concentration of 1 × 10 ⁴ viral particles per cell ( d1312) or a comparable amount of buffer. Then, incubation is carried out at a temperature of 7 ^° C, aimed at ensuring the inclusion of a bound complex of DNA and adenovirus by the cell. The luciferase activity is determined as described above. The results are shown in FIG. 3b.

c) The effect of adenovirus on gene transfer using conjugates of transferrin and polylysine
Conjugate and DNA complexes containing 6 μg pRSVL DNA and 12 μg transferrin and polylysine conjugate (DNA + hTfpL190B) are added to HeLa cells together with an adenovirus at a concentration of 1 • 10 ⁴ viral particles per cell (d1312) or an inactivated heat treatment of d1312 adenovirus at a comparable concentration (d1312 hi). Inactivation by heat treatment is carried out by incubation at a temperature of 45 ^o C for 30 minutes. The luciferase activity is determined as described above. The results of the experiment are presented in FIG. 3s

Example 4. The effect of adenovirus on gene transfer when using conjugates of transferrin and polylysine in selected cell lines. Conjugate and DNA complexes (6 μg pRSVL + 12 μg hTfpL190B) are added to the cells of the cell lines CFT1, KB, HeLa, W138 and MRC5 together with adenovirus d1312 at a concentration of 1 • 10 ⁴ viral particles per cell or without it. Gene transfer efficiency for each cell line is determined as described above by determining luciferase activity. The results of the experiment are presented in FIG. 4.

 Example 5. Improving luciferase gene expression at the level of gene transfer (and not at the level of transactivation). The cell line designated K562 10/6, which constitutionally expresses luciferase, is prepared by transfection with a plasmid containing a fragment of the RSV luciferase gene (Apa1 / Pvu1 fragment of plasmid pRSVL), which is cloned into the CIaI site of the pUC μ locus. This plasmid is transferred into a complex with a conjugate of transferrin and polylysine and K562 cells are transfected with the resulting complex. Since the plasmid of the pUC μ locus contains a neomycin-resistant gene, it is possible to select clones expressing luciferase taking into account neomycin resistance. To carry out further experiments, select clone K562 10/6.

Aliquots of the K562 parent cell line (in 200 μl of RPMI 1640 medium containing 2% TES; 500,000 cells per sample) are treated with either 13 μg TfpL and 6 μg pRSVL or 4 μg pL 90 and 6 μg pRSVL, which are used in 500 μl buffer Hbs. After the addition of adenovirus d1312 in that indicated in FIG. 5 cell concentrations are maintained at a temperature of 37 ^° C. for 90 minutes, after which 2 ml of RPMI medium containing 10% TES is added. Incubation is continued even at a temperature of 37 ^o C for a further 24 hours, after which the cells are prepared to determine the luciferase activity. It was found that incubation in the presence of adenovirus leads to a significant increase in luciferase activity (see Fig. 5A). This applies both to TfpL complexes (2000 light units versus 25000 light units), and to pL90 complexes (0 versus 1.9 • 10 ⁶ light units). This suggests that the K562 cell line has the ability to incorporate pRSVL and polylysine complexes and that this effect is greatly enhanced by the presence of adenovirus, as can be seen from luciferase expression.

Similar experiments were carried out using K561 10/6 cells that constitutionally express the RSVL luciferase gene. In this case, similar amounts of adenovirus d1312 are used. Aliquots of 500,000 cells (in 200 μl of RPMI medium containing 2% TES) are incubated at 37 ^° C for 90 minutes in the presence of adenovirus d1312 in the one indicated in FIG. 5B concentration. Then, as in the case of the parental cell line, RPMI medium containing 10% TES is added, incubation is continued for a further 24 hours, after which luciferase activity is determined. As seen in FIG. 5B, treatment of these cells with an adenovirus does not lead to a noticeable effect on luciferase activity, i.e., the results of control experiments of the same order as the results of experiments that are carried out in the presence of the virus.

Example 6. Transfection of liver cells with conjugates of asialofetuin and polylysine (hereinafter: AfpL) or conjugates of a tetra-galactose peptide and polylysine (hereinafter: gal 4pL) in the presence of adenovirus. a) Obtaining a lactosylated peptide. 3.5 mg (1.82 μmol) of the branched peptide Lys- (N _∈ -Lys) Lys-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Cys containing a dithiopyridine group for Suv, treated with a solution of 7.85 mg of lactose in 40 μl of a 10 mM aqueous solution of sodium acetate with a pH of 5 at a temperature of 37 ^° C. To the solution was added 4 aliquots of 0.6 mg (10 μmol) of sodium cyanoborohydride over a period of about 10 hours. After only 64 hours at a temperature of 37 ^o C add 0.5 ml of HEPES buffer with a pH of 7.3 and 15 mg of dithiothreitol. As a result of fractionation by gel filtration (Sephadex G-10, 12 x 130 mm, eluent: 20 mM sodium chloride) in an argon atmosphere, 3.6 ml of a solution of lactosylated peptide in free mercapto form (1.74 μmol according to Elmann analysis; yield : 84%). Samples of the modified peptide show a color reaction with anisic aldehyde, but no color reaction occurs with ninhydride. This phenomenon is consistent with the assumption that all 4 N-terminal amino groups are lactosylated. The conjugate of the tetra-galactose peptide and polylysine is shown in FIG. 6.

b) Preparation of Modified Polylysine 3-Dithiopyridine Propionate
Intensively stirring 400 μl of a 15 mM ethanolic solution of succinimidyl-thiopyridine propionate (6.0 μmol) is added to a gel-filtered solution of 0.60 μmol of poly-L-lysine in the form of a hydrobromide with an average length of 290 lysine monomers (hereinafter: "pL290") in 1 , 2 ml of 100 mm HEPES buffer with a pH of 7.9. An hour later, 500 μl of 1 M sodium acetate with a pH of 5 was added after gel filtration on Sephadex G-25 using 100 mM sodium acetate. The solution contains 0.56 μmol pL2990 with 5.77 μmol of a dithiopyridine-based binder.

c) The conjugation of the peptide with polylysine
The conjugates are prepared by mixing 1.5 µmol of the lactolized peptide obtained in step a) in 3 ml of 20 mM sodium chloride with 0.146 µl of the modified pL290 obtained in step b) in 620 µl of 100 mM sodium acetate buffer under argon. After adding 100 μl of 2M HEPES pH 7.9 buffer, the reaction mixture was allowed to stand at room temperature for 18 hours. By adding sodium chloride, the salt concentration was adjusted to 0.66 M and the conjugates were isolated by cation exchange chromatography on a HR 5/5 column from Pharmacia, Sweden, using Mono S ion exchanger. Gradient elution was performed using buffer A: 50 mM HEPES with pH 7.3 and buffer B: 50 mM HEPES plus 3 M sodium chloride. Eluting at salt concentrations of about 1.2 M - 1.8 M fractions are collected and combined into two conjugate fractions, designated ga14pL1 and ga14pL2. Dialysis using a 25 mM HEPES buffer at pH 7.3 gives a gal4pLI conjugate containing 24 nmol of modified pL290 and a ga14pL2 conjugate containing 24.5 nmol of modified pL290.

g) Obtaining conjugates of asialofetuin
Conjugates are prepared in substantially the same manner as transferrin conjugates. The binding of asialofetuin to polylysine is carried out through disulfide bridges after modification with succinimidyl pyridyl dithiopropionate. A solution of 100 mg (2.2 μmol) of asialofetuin in 2 ml of 100 mM HEPES pH 7.9 buffer was subjected to gel filtration on a Sephadex G-25 column. To the resulting solution in an amount of 4 ml, stirring vigorously, add 330 μl of a 15 mM methanolic solution of succinimidyl pyridyl dithiopropionate (5.0 μmol). After an hour at room temperature, purification is carried out by gel filtration on Sephadex G-25. In this case, 5 ml of a solution of 1.4 μmol of asialofetuin modified with 2.5 μmol of a dithiopyridine-based binder are obtained.

 Conjugates are prepared by mixing 1.4 μmol of asialofetuin in 5 ml of 100 mM HEPES pH 7.9 with 0.33 μmol of modified pL190 (containing 1.07 μmol of mercaptopropionate groups; proceed in the same manner as for the preparation of transferrin conjugates) 6.5 ml of 200 mm HEPES buffer with a pH of 7.6 in argon atmosphere. The reaction mixture was allowed to stand at room temperature for 24 hours. Conjugates were isolated from the reaction mixture by cation exchange chromatography on an HR 10/10 column using Mono S ion exchanger. Gradient eluvination was performed using buffer A: 50 mM HEPES with pH 7.9 and buffer B: 50 mM HEPES plus 3 M sodium chloride. Sodium chloride is added to the column load until a final concentration of 0.6 M is reached. The target fraction is eluted at a salt concentration of about 1.5 M. Conjugates containing 0.52 μmol of asialofetuin modified with 0.24 μmol are obtained by dialysis using HBS buffer pL190.

e) Transfection of HepG2 cells with pRSVL DNA complexes
HepG2 cells are grown in DMEM containing 10% TES, 100 units of penicillin / ml, 100 μg / ml streptomycin and 2 mM glutamine in T25 flasks. Transfections are carried out at a density of 400,000 cells per flask. Before transfection, the cells are washed with 4 ml of fresh medium containing 10% TES. Immediately prior to transfection, 7-chloro-4 - [(4-dithylamino-1-methylbutyl) amino] -quinoline (hereinafter: “chloroquine”) is added in an amount providing a final concentration in the cell suspension (plus DNA solution) of 100 μM . 10 μg of pRSVL DNA in 330 μl of HBS buffer was mixed with those indicated in FIG. 7 the amount of conjugate of transferrin and polylysine with a molecular weight of 190 (TfpL), conjugate of asialofetuin and polylysine with a molecular weight of 90 (AfpL), polylysine with a molecular weight of 290 (pL) and a conjugate of tetra-galactose peptide and polylysine (ga14pL), which are used in μl of HBS buffer. In competitive experiments, 240 μg of asialofetuin [(gal) 4pL + Af] or 30 μg of the lactosylated peptide [(gal) 4pL + (gal) 4] are used, which are added after 30 minutes. The mixture was added to cells that were incubated at 37 ^° C for 4 hours, after which the transfection medium was replaced with 4 ml of fresh DMEM medium containing 10% TES. After 24 hours, cells are harvested to analyze luciferase activity. In FIG. 7 shows the total luciferase activity in transfected cells. According to the results of the experiments, pL and TfpL show insignificant luciferase activity, (gal) 4pL gives almost the same activity as AfpL, and (gal) 4 and Af, respectively, act as a competitor in the process of binding to asialoglycoprotein and therefore the effect is negligible.

f) Transfection of HepG2 Cells with pCMVL DNA Complexes
HepG2 cells are grown in plates as described in step d), to a density of 300,000 cells per plate. Before transfection, the cells are washed with 1 ml of fresh medium containing 2% TES. 6 μg of pCMVL DNA in HBS buffer was mixed with those indicated in FIG. 8 quantities of conjugate of transferrin and polylysine with a molecular weight of 190 (TfpL), conjugate of asialofetuin and polylysine (AfpL), polylysine with a molecular weight of 290 (pLys290), conjugate (gal) 4pL1 and (gal) 4pL2, which are contained in 170 μl of HBS. After 30 minutes, 1 ml of DMEM medium containing 2% TES and 50 μl of adenovirus d1312C solution are added to each conjugate and DNA complex. As part of competitive experiments, add 30 μg of the lactosylated peptide (gal) 4pL [(gal) 4pL1 + (gal) 4] or [(gal) 4pL2 + (gal) 4]. The mixture is added to the cells, which are incubated at a temperature of 37 ^o C for 2 hours, after which 1.5 ml of medium containing 10% TES are added. After 24 hours, cells are harvested to analyze luciferase activity. Presented in FIG. 8 values represent the total luciferase activity in transfected cells. It can be seen that pLys290 exhibit a certain effect, (gal) 4pL provides an enhanced effect, and the addition of (gal) 4, which competes in the process of binding to asialoglycoprotein, leads to a decrease in the effect achieved by polylysine.

g) Transfection of TIB 73 cells with pCMVL DNA complexes
Cells isolated from the ATCC T1B73 cell line mouse embryonic liver (BNL CL.2) are grown at 37 ^° C. in DMEM containing 0.4% glucose supplemented with 10% inactivated heat treatment of TES containing 100 units / ml penicillin, 100 μg / ml of streptomycin and 2 mm glutamine, in the presence of 5% carbon dioxide. Transfections are carried out at a density of 300,000 cells per dish. Before transfection, the cells are washed with 1 ml of fresh medium containing 2% TES.

6 μg of pCMVL DNA in 300 μl of HBS was mixed with those indicated in FIG. 9 quantities of a conjugate of mouse transferrin and polylysine with a molecular weight of 290 (mTfpL), conjugate of asialofetuin and polylysine (AfpL), polylysine with a molecular weight of 290 (pLys290), conjugate (gal) 4pL1 and conjugate (gal) 4pL2, which are used Hbs. After 30 minutes, 1 ml of DMEM medium containing 2% TES and 50 μl of adenovirus d1312 solution are added to each conjugate and DNA complex. The mixture is added to the cells, which are incubated at a temperature of 37 ^o C for 2 hours, after which 1.5 ml of medium containing 10% TES are added. After two hours, the transfection medium is replaced with 4 ml of fresh medium. After 24 hours, cells are harvested to analyze luciferase activity. In FIG. 9B shows the total luciferase activity in transfected cells.

Comparative experiments are carried out in which transfection is carried out without adenovirus in the presence of chloroquine. The process is also carried out at a density of 300,000 cells per dish. Before transfection, the cells are washed with 1 ml of fresh medium containing 2% TES. Immediately prior to transfection, chloroquine is added in an amount providing a final concentration in the cell suspension (plus DNA solution) of 100 μmol. 6 μg of pCMVL DNA in 330 μl of HBS was mixed with that indicated in FIG. 9In the amount of mTfpL, AfpL, pLys290, (gal) 4pL1 and (gal) 4pL2, which are used in 170 μl of HBS. After 30 minutes, DNA complexes are added to the cells, which are incubated at a temperature of 37 ^o C for 2 hours, after which 1.5 ml of medium containing 10% TES and 100 μmol of chloroquine are added. After two hours, the transfection medium is replaced with 4 ml of fresh medium. After 24 hours, cells are harvested to analyze luciferase activity. The results of the experiments are presented in FIG. 9A.

Example 7. The introduction of DNA into T cells
a) Obtaining conjugates of antiCD7 and polylysine with a molecular weight of 190
A solution of 1.3 mg of antiCD7 antibody in 50 mM HEPES pH 7.9 buffer was mixed with 49 μl of a 1 mM ethanolic solution of succinimidyl pyridyl dithiopropionate. After one hour at room temperature, the mixture was subjected to gel filtration on a Sephadex G-25 column using 50 mM HEPES pH 7.9 buffer as eluent.

 1.19 mg (7.5 nmol) of anti-CD7 modified with 33 nmol of pyridyldithiopropionate groups are obtained. A poly (L) lysine with a molecular weight of 190, labeled with a fluorescent agent, is similarly modified with succinimidyl pyridyl dithiopropionate and converted to the free mercapto group modified form by treatment with dithiothreitol followed by gel filtration. A solution of 11 nmol of polylysine with a molecular weight of 190, modified with 35 nmol of mercapto groups, in 0.2 ml of 30 mM sodium acetate buffer is mixed with modified antiCD7 in 0.5 ml of 300 mM HEPES buffer with pH 7.9 under conditions of oxygen exclusion and the mixture left to stand overnight at room temperature. The reaction mixture was adjusted to a concentration of about 0.6 M by adding 5 M sodium chloride. The conjugates were isolated by ion exchange chromatography on Mono S ionite (eluent: 50 mM HEPES buffer, pH 7.3, saline gradient: 0.6-3 M sodium chloride). After dialysis with a 10 mM HEPES pH 7.3 buffer, a conjugate of 0.51 mg (3.2 nmol) of anti-CD7 antibody modified with 6.2 nmol of polylysine with a molecular weight of 190 is obtained.

 b) Obtaining conjugates of gp120 and polylysine with a molecular weight of 190.

 Binding is carried out via thioether groups after modification with 6-maleimidocaproic acid N-hydroxy-succinimide ester. The process is as follows.

 A solution of 2 mg of recombinant gp120 in 0.45 ml of 100 mM HEPES pH 7.9 buffer was mixed with 17 μl of a 10 mM solution of 6-maleimidocaproic acid N-oxysuccinimide ester in dimethylformamide. After one hour at room temperature, gel filtration was performed on a Sephadex G-25 column using 100 mM HEPES buffer, pH 7.9, as eluent. Obtain 1.2 ml of the target solution, which, under conditions of exclusion of oxygen, is immediately reacted with a solution of 9.3 nmol of polylysine with a molecular weight of 190, labeled with a fluorescent agent and a modified 30 nmol of mercapto groups in 90 μl of 30 mm sodium acetate with a pH of 5.0 . The reaction mixture was left to stand overnight at room temperature, after which its concentration was adjusted to approximately 0.6 M by the addition of 5 M sodium chloride. The conjugates were isolated by ion exchange chromatography on a Mono S column using 50 mM HEPES pH 7.3 (eluent: 0.6 - 3 M sodium chloride) as eluent. After fractionation and dialysis using 25 mM HEPES buffer at pH 7.3, conjugates A, B and C of the following composition are obtained. Conjugate A consists of 0.40 mg of gp120, modified 1.9 nmol of polylysine 190, conjugate B is 0.25 mg of gp120, modified 2.5 nmol of polylysine 190, and conjugate B is 0.1 mg of gp120, modified 1.6 nmol polylysine 190.

6 μg pCMVL DNA per sample was transferred to a complex with polylysine 190 or its conjugates in 500 μl HBS. The obtained complexes of polylysine and DNA are added to H9 cells (10 ⁶ cells in 5 ml of RPMI medium containing 2% DES) or to primary human lymphocytes (3 • 10 ⁶ cells in IMDM medium containing 2% DES). After 5 minutes, d1312 adenovirus is added in the amount indicated in FIG. 10. Cells were incubated at 37 ^° C for 90 minutes, after which 15 ml of RPMI medium (in the case of H9 cells) or IMDM (in the case of primary lymphocytes) and 20% DES were added to each sample. Cells are incubated at a temperature of 37 ^o C for only 24 hours, after which they are collected for analysis of luciferase activity. The results of the experiments are presented in FIG. 10 A (in the case of H9 cells) and FIG. 10 V (in the case of primary lymphocytes). In FIG. 10A, the anti-CD7 conjugate (traces 7-9) and the gp120 conjugate (traces 10-12) show the best results with respect to the gene transfer achieved by adenovirus, while the gp120 conjugate exhibits clear luciferase gene expression even in the absence of adenovirus. It should also be noted that only the conjugate gp120 is capable of introducing DNA into primary lymphocytes under the condition of the presence of adenovirus (see Fig. 10 B, traces 7.8).

Example 8. Inactivation of adenoviruses
a) Inactivation by UV radiation.

Obtained in the above manner, the preparation of adenovirus d1312 is fed into a cup equipped with cells with a diameter of 2 cm (300 μl per well). At a distance of 8 cm from the cup, two UV lamps of the G15 T8 brand of a foreign company Philips are installed. The virus is irradiated with ultraviolet rays during the period shown in Fig. 11A time. Aliquots of each drug are examined for the titer of the virus and determine the ability to improve gene transfer into gene cells when using conjugates of polylysine and transferrin. Cell cultivation and transfection is carried out mainly as described above. The components used for transfection are shown in FIG. 11A. In 500 μl of HBS buffer, pCMVL DNA complexes and 12 μg of TfpL are obtained, which are added to 3 • 10 ⁵ HeLa cells in 1 ml of DMEM medium containing 2% TES. After 5 minutes, 54 μl of each virus preparation was added to each culture, and the culture was incubated at 37 ^° C. for 60-120 hours. Then, a 5 ml aliquot of DMEM medium containing 10% TES is added to each culture, incubation is continued at 37 ^° C for 24 hours, after which cultures are collected for luciferase activity studies. The amount of 54 μl of unirradiated virus is not within the saturation limits, that is, the experiment is sensitive to the amount of virus that exceeds the specified at least 3 times. The results obtained for luciferase expression are shown in FIG. 11 B (dark rectangles). The virus titer of each preparation is determined using an Ela-compliant cell line 293. In a DMEM medium containing 2% TES, a number of diluted groups of irradiated and unirradiated virus are prepared. In parallel, in 200 μl of DMEM medium containing 2% TES, samples of 5 • 10 ⁴ 293 cells were prepared (using a cup with cells with a diameter of 2 cm). An aliquot of 5 μl of each diluted sample is supplied to each well. In order to achieve the binding of the virus to the cells, incubation is carried out at a temperature of 37 ^° C for 90 minutes, after which 2 ml of DMEM medium containing 10% TES is fed into each cell. After 48 hours, cultures are examined for the degree of cell disease. A diluted virus sample with less than 50% of the cells in the culture, showing a significant degree of disease after 48 hours, indicates the relative amount of the infectious virus in each preparation. The results are presented in FIG. 11 B (open rectangles). The results of the experiment of this example show that the decrease in the titer of the virus by 4 log caused by UV radiation is associated only with a 20-fold decrease in the degree of luciferase gene transfer. Thus, the mechanisms crucial for virus infectivity can be eliminated without a significant negative effect on the ability of the virus to improve gene transfer. It was found that in small doses of the virus, the improvement in gene transfer provided by the virus decreases slightly (see FIG. 11A, traces 3-6), and that this effect is more pronounced at high concentrations (see traces 7-10).

 b) Inactivation of adenoviruses with formaldehyde.

2 ml of the adenovirus preparation is passed through a 10 ml capacity Sephadex G-25 column, which is pre-equilibrated with 150 mM sodium chloride, 25 mM HEPES buffer with a pH of 7.9 and 10% glycerol. Aliquots of the gel-filtered virus preparation are incubated on ice for 20 hours in the presence of 0.01%, 0.1% and 1% formaldehyde. In addition, conduct a control experiment without the use of formaldehyde. Tris buffer with a pH of 7.4 is added until a concentration of 100 mM is established, after which the samples are subjected to two-stage dialysis, with 1 liter of 150 mM sodium chloride, 50 mM Tris with a pH of 7.4 and 50% glycerol for two hours and in the second stage, dialysis is performed overnight using 2 • 1 L of 150 mM sodium chloride, 20 mM HEPES with a pH of 7.9 and 50% glycerol. Aliquots of the virus are then tested for titer using 293 cells. Then, the effect of the formaldehyde-treated virus on gene transfer to HeLa cells (300,000) is determined by measuring luciferase activity in the above manner. Treatment of the virus with formaldehyde in an amount of 0.01% and 0.1% leads to a slight decrease in gene transfer activity (approximately 10-fold decrease at 0.1%). Although treatment with 1% formaldehyde causes a significant loss in gene transfer activity, 90 μl of the virus still exhibits the ability to express a gene corresponding to 10 ⁴ light units. The decrease in the titer of the virus to 10 ⁵ plaque-forming units observed during processing of 0.1% formaldehyde is associated with a decrease in luciferase activity by only 10%. The results of this experiment are presented in Fig.12 A.

 c) Inactivation of adenoviruses by long-wave UV irradiation and treatment with 8-methoxy-psoralen (psoralen = 7H-furo [3,3-g] [1] benzopyran-7-one).

 Aliquots of the purified virus are added to 0.33 μg / μl of 8-methoxy-psoralen in dimethyl sulfoxide and exposed to a 365 nm UV light source located at 4 cm from the samples on ice. Irradiation is carried out for 15 to 30 minutes. Virus samples are then passed through a Sephadex G-25 column, which is pre-equilibrated with HBS buffer and 40% glycerol. The viral preparations are then examined for their ability to improve the transfer of the pCMVL complex and hTfpL conjugate to HeLa cells by determining the light units of luciferase activity, as well as their ability to replicate in 293 cells by determining the titer. The results of the experiment are presented in Fig.12 Century

 In the following examples, which illustrate the improvement in cell uptake of complexes of DNA and the conjugate of transferrin and polylysine in the presence of retroviruses, the following materials and methods are used.

Conjugates of transferrin and polylysine with a molecular weight of 190 and complexes of the conjugate and DNA are prepared as described above. NIH3T3 cells were grown in DMEM supplemented with 10% TES, 100 units / ml penicillin, 100 μg / ml streptomycin and 2 mM glutamine. Cells are grown in an amount of 5 - 7 • 10 ⁵ for each sample 18-24 hours before transfection. Immediately before transfection, the cells are fed into fresh medium, after which they add successively: 100 μmol (if nothing else is indicated) of chloroquine, a complex of DNA and conjugate of polylysine and transferrin, and a retrovirus preparation. Then the cells are incubated at a temperature of 37 ^o C for 4 hours, after which the medium is replaced and after 24 hours the cells are harvested. The extracts are carried out in three cycles of freezing and thawing. After standardization with respect to protein content, aliquots of the extracts are examined for luciferase activity in the above manner.

Example 9. Transfection of NIH3T3 Cells with Molonea Virus
10 ⁶ NIH3T3 cells are transfected with a complex of DNA and TfpL conjugate in the presence or absence of 100 μmol of chloroquine. The experimental results are presented in Fig. 13. It was found that without the use of chloroquine, the luciferase activity values only reach the background level (trace 1), while in the presence of chloroquine, high expression of the pRSVL reporter gene can be determined (see trace 2). The increasing amounts of the Molonea leukemia virus (indicated by RVS in Fig. 13), which are added to the cells simultaneously with this complex, lead to further improvement in the expression of the luciferase gene.

Example 10. The effect of Molonea virus on gene transfer in the case of transfection of NIH3T3 cells with a complex of DNA and conjugate of transferrin and polylysine. The virus preparations used in Example 9 are a crude, non-fractionated supernatant of cells expressing a retrovirus. To confirm that the improvement in DNA transfer achieved in the presence of the virus preparation can indeed be attributed to the virus, the supernatant is purified by dialysis and concentration on factor 10. If the retrovirus is responsible for the improvement, then the membrane-retained activity (despite any inactivation of the highly unstable retrovirus during concentration ) should exceed the activity of the initial supernatant by about 10 times. As in the previous example, 10 ⁶ NIH3T3 cells were transfected under the conditions shown in FIG. 14. In FIG. 14 it can be seen that the gene transfer improving effect is characteristic of the membrane retention product (20-600 μl are used, traces 3-6). In addition, it was found that 200 and 600 μl of a 10-fold concentrated preparation show approximately half the activity of 2 and 6 ml of the initial, i.e., non-concentrated preparation of retrovirus (traces 7, 8). Parallel experiments are also carried out using human K 562 cells that do not have a receptor for an ecotropic mouse retrovirus. As expected, no improvement in gene expression was observed.

Example 11. The interaction between transferrin and its receptor plays a role in gene transfer, which is promoted by the Molonea virus
The retrovirus is examined for the ability to transfer plasmid DNA into a cell in the form of a complex with one polylysine in order to clarify the issue of attributing the transfer of the pRSVL complex and TfpL conjugate to cells to the non-specific connection of polylysine with retrovirus, and to further study the mechanism of entry into the cell. The amount of polylysine used corresponds to the optimal amount, which ensures complete condensation of plasmid DNA. The experiments, the results of which are presented in FIG. 15 show that in the absence of chloroquine the reporter gene is not expressed either with the use of pRSVL and TfpL conjugate complexes or with the use of pRSVL and polylysine complex (see traces 1, 2). However, in the presence of a retrovirus, reporter DNA, which is used as a complex with the TfpL conjugate, is expressed, while expression of the DNA and polylysine complex is not observed (see traces 3, 4 compared to traces 5, 6). In addition, these experiments show that the presence of excess free transferrin leads to a decrease in the DNA transfer achieved by the retrovirus (see traces 7, 8). These results reinforce the assumption that the interaction between transferrin and its receptor plays a significant role in improving the uptake of DNA provided by the retrovirus.

 Example 12. The effect of pH on the retrovirus gene transfer. The experiments of this example are aimed at studying the effect of pH on the ability of retroviruses to improve gene transfer. In these experiments, transfection is carried out as described above. Two well-known nucleolus pH lowering inhibitors, i.e. monensin and ammonium chloride, are used. The results of the experiments are presented in FIG. 16. A study of the effect of both substances on the transfer of the complex of DNA and the TfpL conjugate shows that, functionally, neither of the two substances can replace chloroquine. However, at higher concentrations of ammonium chloride, there is a slight improvement in the expression of the luciferase gene (see tracks 1 to 5). One retrovirus shows a slight improvement in DNA transfer, as observed in the previous examples (see trace 6). A sharp improvement is observed with the use of retrovirus in the presence of 1 μmol of monensin (see trace 7). A less strong effect is observed at a higher concentration of monensin (see trace 8), as well as in the presence of ammonium chloride (see tracks 9, 10).

Example 13. Improvement in gene transfer achieved by conjugates of transferrin in the presence of the N-terminal endosomolytic peptide of hemagglutinin HA2 influenza
a) Peptide synthesis
The peptide with the sequence:
Gly-Leu-Phe-Glu-Ala-Ile-Ala-Gly-Phe-Ile-Glu-Asn-Gly-Trp-Glu-Gly-Met-Ile-Asp-Gly-Gly-Gly-Cys are synthesized by known methods using fluorenylmethoxycarbonyl. Tertiary butyl (for Cys, Glu and Asp) and trityl for Asn are used as side-chain protecting groups. After the reaction, the degree of binding using ninhydrin is analyzed. In this case, a degree of> 98% is established at each stage. Starting from amino acid 19, a double combination is performed. N-terminal fluorenylmethoxycarbonyl groups are removed from a portion of the peptide resin by treatment with 20% piperidine in N-methylpyrrolidine. Then the fractions protected by fluorenylmethoxycarbonyl and the unprotected fractions are washed with dichloromethane and dried under high vacuum. 294 mg of a fluorenylmethoxycarbonyl-free peptide resin and 367 mg of a fluorenylmethoxycarbonyl-protected peptide are obtained. 111 mg of the fluorenylmethoxycarbonyl group-free peptide resin is cleaved by treatment with a mixture of 10 ml of trifluoroacetic acid, 0.75 g of phenol, 300 μl of ethanedithiol, 250 μl of ethyl methyl sulfide and 500 μl of water for 90 minutes. The peptide is separated from the resin by filtration. The resin is washed with dichloromethane and added to the filtrate, which is concentrated to a volume of about 2 ml, after which, with stirring, it is added dropwise to 40 ml of diethyl ether. The peptide precipitate was removed by centrifugation and the ether supernatant was removed. The precipitate was washed three times with 40 ml of diethyl ether and dried in high vacuum. Obtain 58 mg of crude product, which is dissolved in 3.5 ml of 20 mm ammonium bicarbonate containing 300 μl of 25% ammonia / L. The solution was subjected to gel filtration on a Sephadex G-25 column using the same buffer. Then the eluate is fed to a Mono Q brand column measuring 100 x 14 mm, in which the process is carried out under the following conditions. Gradient: 0-10 minutes: 100% A, 10-100 minutes: 0-100% B, with A = 20 mM ammonium bicarbonate + 300 μl 25% ammonia / l, aB = A + 3M sodium chloride. Measured at 280 nm, and Trp fluorescence detection at 354 nm. Delivery rate: 1 ml / min. The product is eluted with 1 M sodium chloride. The main column fraction is further purified by reverse phase liquid chromatography, which is carried out under pressure in a BIORAD-Hi-Pore RP 304 column of size 250 x 10 mm. The following conditions are observed. Gradient: 50-100% of buffer A for 12.5 minutes, 12.5 - 25 minutes: 100% B; wherein A = 20 mM ammonium bicarbonate + 300 μl 25% ammonia / l, and B = A in 98% methanol. Delivery rate: 3 ml / min. Measured at 237 nm. The product is eluted at 100% B.

 The desired fractions were evaporated, redissolved in buffer A and then lyophilized. 8.4 mg of a purified cysteine-protected liquid chromatography product is obtained. This peptide is designated P16. In order to obtain P16 in a free mercapto form, a tertiary butyl protected substance is treated with a mixture of thioanisole, ethanediol, trifluoroacetic acid and trifluoromethanesulfonic acid in a ratio of 2: 1: 40: 3 (trifluoromethanesulfonic acid is added as the last component) at room temperature for 30 minutes. The peptide is isolated by precipitation with diethyl ether and subsequent gel filtration on Sephadex G-25 using the aforementioned buffer A in an argon atmosphere.

b) The binding of the peptide of influenza with polylysine
b1) Direct binding with succinimidyl pyridyl dithiopropionate
19.8 mg of polylysine hydrobromide with a molecular weight of 300 is subjected to gel filtration on a Sephadex G-25 column using a solution of sodium acetate with a pH of 5, aimed at removing low molecular weight fractions. According to the analysis using ninhydrin after gel filtration, the concentration of polylysine is 3.16 mg / ml. By adding 1 M sodium hydroxide, the pH of the solution was adjusted to 7-8. To 2.5 ml of a solution of polylysine (polylysine content of 7.9 mg = 0.13 μmol) was added 0.64 μmol of succinimidyl pyridyl dithiopropionate in the form of a 40 mM solution in absolute ethanol. Thus, the molar ratio of succinimidyl pyridyl dithiopropionate and polylysine is 5: 1. The mixture was allowed to react overnight, after which it was subjected to gel filtration on a Sephadex G-25 column using 20 mM ammonium bicarbonate with a pH of 8.2. After restoration of an aliquot of the filtrate with dithiothreitol, the thiopyridone content is determined. According to the data of this analysis, the reaction proceeded completely. 0.3 μmol of the modified polylysine pyridyldithiopropionate (calculated as μmol of pyridyldithiopropionate) is reacted with 0.35 μmol of the peptide in thiol form. The white precipitate obtained by mixing the peptide and polylysine is dissolved in 2 M guanidinium hydrochloride. The reaction mixture was left to stand overnight, after which a photometric determination of the content of thiopyridone in the reaction mixture was carried out. The data from this analysis confirm the completeness of the reaction. The mixture is then dialyzed twice with 2 L of 20 mM HEPES buffer containing 0.5 M guanidinium hydrochloride. The resulting solution is fed to a Mono S brand column measuring 0.7 x 6 cm. The following conditions are observed. Gradient: 0 - 20 min: 100% A, 20-140 min: 0-100% B, where A means 20 mM HEPES with a pH of 7.3 / 0.5 M guanidinium hydrochloride, and C - 20 mM HEPES with a pH of 7 , 3 plus 3 M guanidinium hydrochloride. Delivery rate: 0.3 ml / min. Detection at 280 nm, fluorescence detection at 354 nm, and excitation at 280 nm. The target fraction was eluted with 1.5 M guanidinium hydrochloride followed by dialysis using 2 L of HBS. According to the subsequent determination of the concentration of polylysine using ninhydrin, its concentration is approximately 1.14 mg / ml. The amount of peptide in the conjugate solution is calculated taking into account its absorption at 280 nm. According to these data, the molar ratio of peptide to polylysine is 4: 1.

b2) Binding using a polyethylene glycol-based binder
14.6 mg of polylysine hydrobromide with a molecular weight of 300 is subjected to gel filtration similarly to step b1). According to the experience with ninhydrin, the concentration of polylysine after gel filtration is 4.93 mg / ml. By adding 1 M sodium hydroxide, the pH of the solution was adjusted to 7-8. To 2.7 ml of a solution of polylysine (polylysine content of 13.3 mg = 0.22 μmol), 4.33 μmol of pyridyldithiopropionate is added as a 30 mM solution in absolute ethanol. Thus, the molar ratio of pyridyldithiopropionate to polylysine is 20: 1. After 90 minutes, the reaction mixture was subjected to gel filtration on a Sephadex G-25 column using 0.1 M sodium acetate and 3M guanidinium hydrochloride. After restoration of an aliquot of the filtrate with dithiothreitol, the thiopyridone content is determined. According to this analysis, the target fraction contains 3.62 μmol of pyridyldithiopropionate. Modified pyridyldithiopropionate polylysine is subjected to restoration used in the amount of 79 mg of dithiothreitol for 2 hours. The solution was again filtered on a G-25 column under the indicated conditions. According to the Elman method, the thiol concentration is 3.15 μmol in 2.224 ml.

 17.6 mg (5 μmol) of polyoxyethylene bis (6-aminohexyl) are dissolved in 500 μl of 20 mM sodium bicarbonate and 3 M guanidinium hydrochloride with a pH of 7-8, after which they are reacted with 13.8 mg of ε-N-oxysuccinimide ester - maleimido-caproic acid (= 44.6 μmol) dissolved in 300 μl of dimethylformamide. After 30 minutes, the solution is subjected to gel filtration on a G-25 column using 20 mM sodium bicarbonate and 3 M guanidinium hydrochloride. According to the photometric determination of the maleimido group at 300 nm, the concentration of the reacted polyoxyethylene bis (6-aminohexyl) is 6.36 μmol in 2 ml of solution. To 1.05 ml of this solution (corresponding to 3.34 μmol of the indicated ether) 1.39 μmol of peptide in thiol form in 2.5 ml of 20 mM sodium bicarbonate and 3 M guanidinium hydrochloride are added dropwise. The mixture is intensively stirred in an argon atmosphere. After 15 minutes, free thiol groups can no longer be detected.

The reconstituted mercapto-modified polylysine solution was adjusted to pH 7-8 by adding 1 M sodium hydroxide. 1.37 ml of this solution, stirring vigorously, was added to the above reaction mixture. Thus, the molar ratio of peptide-SH, N-oxysuccinimide ester of ε-maleimido-caproic acid and polylysine-SH is 1: 2.4: 1.4 (based on said ether and SH). Free thiol groups can no longer be detected after 150 minutes. The reaction mixture was dialyzed overnight using 2 L of 20 mM HEPES with a pH of 7.3 plus 0.6 M sodium chloride, and then fed to a Mono S column. The process was carried out under the following conditions. Gradient: 1 ml of the aqueous phase (10 mM HEPES with a pH of 7.3; 100 mM sodium salt of fluorescein-2,7-bis-methylene iminodiacetic acid (hereinafter: calcein); 150 mM sodium chloride) are washed thoroughly with the ether phase and sonicated at a temperature 0 ^o C for 5 minutes. After 30 minutes on ice, the material is again sonicated for a further 10 minutes. The resulting stable emulsion is slowly evaporated. After removal of diethyl ether at 100 mbar, 0.75 ml of the aforementioned aqueous phase is added. The remainder of diethyl ether is removed by further evaporation at a pressure of 50 mbar for 30 minutes. 1.7 ml of residue are obtained, which is centrifuged at 500 rpm. 1.0 ml of the centrifugation product is passed through a microporous polycarbonate membrane with a pore size of 0.1 μm to obtain 0.7 ml of liposome solution. Liposomes are separated from unincorporated material by gel filtration (Sephadex G-50 medium from Pharmacia, Sweden; 23 ml of gel volume, 10 mM HEPES with pH 7.3 / 150 mM sodium chloride). Collect 6 fractions with a volume of 500 μl. Lipid phosphorus is determined at 2 mm.

g) Determination of release from liposome contents
Isolation from liposomes is determined by measuring the release of the enclosed calcein and the resulting dilution, which stops the quenching of fluorescence. Calcein fluorescence is determined using a Kontron SMF 25 spectral fluorometer (excitation at 490 nm, emission at 515 nm). For this purpose, 100 μl aliquots of the above liposome solution are diluted 100 times with 0.1 M sodium acetate / 50 mm sodium chloride or 10 mm NEPES / 150 mm sodium chloride with an appropriate pH value (4.3, 4.5, 5.0, 6 , 7.3). To the resulting 1 ml solution was added 2.5 μl of the peptide in tertiary butyl protected form (1 μg / μl of the solution in HBS) while mixing under argon atmosphere (final concentration: 400 nM peptide). Calcein fluorescence is determined at different times after the addition of the peptide. Values for 100% isolation are determined by adding 2 μl of X-100 Triton. The same techniques are used to determine the fluorescence of calcein after adding peptide and polylysine conjugates to the liposome solution. At the same time, 2.5 μg of conjugate is added to 1 ml of liposome solution (1 μg / μl of concentration in terms of the amount of one polylysine). In this case, the final concentration is 20 nm of the modified peptide. In a similar manner, 2.5 μg of the conjugate of the peptide and polylysine after incubation together with 5 μg of DNA for 15 minutes is subjected to analysis. It was found that the peptide causes the release of liposome contents only in the acidic pH region (see Fig. 17). The peptide conjugate is active at a significantly lower pH, although significant activity is observed even at a neutral pH, which further increases as the pH decreases. The conjugate-DNA complex abolishes activity at a neutral pH, while at acidic pH a marked activity is observed. In FIG. 17 peptide designated Influ.

d) Transfection of K562 cells
K562 cells are grown in suspension in RPMI 1640 medium (plus 2 g sodium bicarbonate / l, 10% TES, 100 units / ml penicillin, 100 μg / ml streptomycin and 2 mM glutamine) until a density of 500,000 cells / ml is reached. 12 to 20 hours before transfection, the cells are fed into fresh medium containing 50 μM desferrioxamine, 30-amino-3.14.25-trioxy-3.9.14.20.25-pentaazatriacontan-2.10.13.21.24-pentaon (in order to increase the number of transferrin receptors ) On the day of transfection, cells are harvested, suspended in fresh medium containing 10% TES and 50 μm desferrioxamine (250,000 cells per ml) and served in 2 ml portions in a cup equipped with 24 cells. 6 μg of pCMVL DNA in 160 μl of HBS was mixed with that indicated in FIG. 18 by the amount of TfpL conjugate or with pL300 in 160 μl of HBS, after 15 minutes the indicated quantities of the influenza peptide and polylysine conjugate (P16pL) are added and after a further 15 minutes the mixture is added to K562 cells, which are incubated at 37 ^° C for 24 hours, after whereupon they are collected to determine luciferase activity as described in the previous examples. Presented in FIG. 18 values represent the total luciferase activity of transfected cells.

e) Transfection of HeLa cells
HeLa cells are cultured in 6 cm diameter dishes as described above. Transfection is carried out at a density of 300,000 cells per dish. Before transfection, cells are incubated in 1 ml of fresh medium containing 2% TES. 6 μg of pCMVL DNA in 160 μl of HBS was mixed with that indicated in FIG. 19 by the amount of TfpL conjugate or polylysine with a molecular weight of 300 (pL300) or a mixture of pL300 conjugate in 160 μl of HBS. After 15 minutes, the indicated amounts of the influenza peptide conjugate and pL (P16pL) were added and after a further 15 minutes the mixture was added to cells that were incubated at 37 ^° C for 2 hours, after which 2.5 ml of fresh medium containing 10% TES was added. . Cells are incubated at a temperature of 37 ^o C for 24 hours, after which they are collected to determine the activity of luciferase as described in the previous examples. Presented in FIG. 19 values represent the total luciferase activity of transfected cells.

Example 14. The improvement in gene transfer achieved by conjugates of transferrin using the second N-terminal endosomolytic peptide of hemagglutinin HA2 influenza
a) Synthesis of the conjugate of the peptide of influenza and polylysine
Peptide by the sequence Gly-Leu-Phe-Gly-Ala-Ile-Ala-Gly-Phe-Ile-Glu-Asn-Gly-Trp-Glu-Gly-Met-Ile-Asp-Gly-Gly-Gly-Cys (designated P41 ) are synthesized by a known method. The binding of the influenza peptide to polylysine (pL300) is carried out in the same manner as in example 13, b1), i.e. using succinimidyl pyridyldithiopropionate. Conjugates (P41pL) are obtained in which the molar ratio of peptide to polylysine is 4: 1.

b) Transfection of HeLa cells
HeLa cells are grown in DMEM containing 5% TES, 100 units / ml penicillin, 100 μg / ml streptomycin and 2 mM glutamine, which is carried out in 6 cm diameter dishes. Transfection is carried out at a density of 300,000 cells per dish. Before transfection, cells are incubated in 165 ml of fresh medium containing 2% TES. 6 μg of pCMVL DNA in 160 μl of HBS (150 mM sodium chloride, 20 mM HEPES with a pH of 7.3) was mixed with 6 μg of TfpL190B conjugate in 160 μl of HBS, after 15 minutes 10 μg of P41pL conjugate was added or, as a comparison, 18 μg P16pL conjugate (see Example 13). The indicated amounts of both peptide-containing conjugates are optimal to achieve improved gene transfer. After a further 15 minutes, the mixture was added to cells that were incubated at 37 ^° C for 4 hours, after which 2 ml of medium containing 18% TES was added. After 24 hours, cells are harvested to determine luciferase activity. Presented in FIG. 20 values represent the total luciferase activity of transfected cells. A comparison of the results obtained using both peptide-containing conjugates shows that the P41pL conjugate provides an improvement in gene transfer, which is 3.5 times higher than the result achieved by the other peptide-containing conjugate.

c) Transfection of BNL CL.2 cells with peptide conjugates of influenza virus and polylysine
BNL CL.2 cells are grown analogously to Example 6. P41 peptide is conjugated to polylysine with a molecular weight of 300 at a molar ratio of 1: 1, 3: 1 and 8: 1, respectively. Complexes of 6 μg pCMVL DNA and 20 μg conjugates are added to the cells. As a comparison, 20 μg of polylysine with a molecular weight of 300 (pL300) or 20 μg of P16 conjugate according to Example 13 are used. The cells are incubated at 37 ^° C for 4 hours, after which 2 ml of medium containing 18% TES are added. After 24 hours, cells are harvested to determine luciferase activity. The results of the experiment are presented in FIG. 20V. The peptide content in the conjugates is correlated with improved gene expression. According to the analysis of the isolation of contents from liposomes (see Fig. 20C), which is carried out as described in Example 13, the activity of conjugates (at pH 5, which is equivalent to 2.5 μg of polylysine) increases as the content of the peptide increases. In FIG. The 20 C peptide P41 is designated "influ2".

Example 15. Transfection of HeLa cells with a β-galactosidase reporter gene construct and in situ expression of β galactosidase expression
a) Cell growth and transfection
HeLa cells are grown in the same way as in the previous examples in DMEM containing 5% TES, penicillin, streptomycin and glutamine, the process being carried out in 3 cm diameter dishes. Density: 3 • 10 ⁴ cells per dish.

 6 μg of β-galactosidase reporter gene construct (hereinafter: pCMV-β-gal) in 160 μl of HBS was complexed with 12 μg of TfpL190B in 160 μl of HBS by incubation at room temperature for 30 minutes. In addition, 6 μg of pCMV-β-gal in 160 μl of HBS is incubated with 6 μg of TfpL190B in 80 μl of HBS at room temperature for 15 minutes. Then add 12 μg of the conjugate of the influenza peptide (P16pL) according to example 13 in 80 μl of HBS and the mixture is incubated for 15 minutes. The resulting complexes of DNA and polycation are mixed with 1 ml of DMEM medium containing 2% TES, antibiotics and glutamine, as described above. In order to identify the effect of chloroquine and adenovirus on the success of transfection, additional experiments are carried out using chloroquine at a final concentration of 100 μM and a solution of adenovirus strain d1312 at a final concentration of 50 μl, which is added to the medium containing DNA and polycation complexes.

Before transfection, the initial nutrient medium is removed and 1 ml of medium containing DNA complexes and chloroquine or adenovirus, or 1 ml of medium that contains only DNA complexes is added to the cells. After two hours of incubation at 37 ^° C, 1 ml of DMEM medium containing 10% TES, antibiotics and glutamine are added to the cells, and the incubation process is continued for a further 2 hours. Then the whole medium is removed and the cells are cultured in 3 ml of fresh DMEM medium containing 10% TES, antibiotics and glutamine.

b) Determination of express β-galactosidase
48 hours after transfection, the medium is removed, the cells are washed with phosphate-containing saline (hereinafter: PBS buffer) and subjected to interaction with 0.5% glutardialdehyde in PBS buffer at room temperature for 5 minutes. Then, the unreacted dialdehyde is removed and the cells are washed with PBS, followed by incubation with a staining solution (10 mM phosphate buffer, pH 7.0, 150 mM sodium chloride, 1 mM magnesium chloride, 3.3 mM K ₄ Fe (CN) ₆ • 3H ₂ O, 3.3 mM K ₃ Fe (CN) ₆ and 0.2% 5-bromo-4-chloro-3-indolyl-β-galactopyranoside) at a temperature of 37 ^o C for 20 minutes up to 3 hours. Then the cup lids are washed with PBS, water and 96% ethanol. After drying, analysis is carried out using a Zeiss axiofot microscope. In FIG. 21 presents photographs of microscopic magnifications (112 times). A: HeLa cells transfected with a complex of 6 μg pCMV-β gal and 12 μg TfpL190B. The staining reaction is carried out for 3 hours. In FIG. it is seen that only a small number of cells (55 cells; the group of stained cells is indicated by an arrow) expresses the β-galactosidase gene. B: HeLa cells transfected with a complex of 6 μg pCMV-β-gal, 6 μg TfpL190B and 12 μg P16pL. The staining reaction is also carried out for 3 hours. A small number of cells (250) express the β-galactosidase gene. However, the cell response is stronger than in case of A. C: HeLa cells transfected with a complex of 6 μg pCMV-β-gal, 6 μg TfpL190B and 12 μg P16pL in the presence of 100 μM chloroquine. In this case, the staining reaction is also carried out for 3 hours. A large number of cells exhibits a significant positive reaction (more than 1000 cells). D: HeLa cells transfected with a complex of 6 μg pCMV-β-gal and 12 μg TfpL190B in the presence of adenovirus d1312. The staining reaction is carried out for 20 minutes. Almost all cells (more than 90%) show a positive reaction. E: Non-transfected HeLa cells (control experiment to determine the specificity of the β-galactosidase reaction). In this case, the staining reaction is carried out for 3 hours.

Example 16. Transfection of HeLa cells with a cosmid of 48 kbp size in the presence of adenovirus
a) Obtaining a cosmid containing a sequence encoding a luciferase
The 3.0 kb SalI fragment containing the P. pyralis luciferase coding sequence under the control of the RSV promoter was isolated from plasmid p220RSVLuc α and doped into the SAlI site of the cosmid clone C1-7aA1 in order to form concatemers. The cosmid clone C1-7aA1 comprises a Sau3A fragment of human genomic DNA with a size of 37 kbp (partial digestion), which does not encode specific genes and is cloned into the BamHI site of the cosmid vector pWE15. The doping products are packaged in vitro, an aliquot of the resulting phage particles is sent for infection in E. coli NM544 and served on LB amp plates. Recombinant products are screened by colony hybridization using a 3.0 kp SalI fragment as a hybridization sample. about. labeled with ³² P. A number of positive products are detected by restriction mapping. The cosmid construct (CosLuc), including a single copy of the SalI insert, is grown and purified on a cesium chloride gradient (total size: 48 cbp).

 A small control cosmid pWELuc measuring 12 kbp obtained by digesting CosLuc using NotI, re-doping, transforming bacteria and isolating a clone containing the target plasmid. A 12 kbp DNA molecule is obtained which is insufficient by inserting human DNA and part of the CosLuc polylinker. Plasmid pSPNeoLuc 8 kbp is a plasmid described in Example 5 that contains a fragment of the RSV luciferase gene (Apa1 / Pvu1 fragment of pRSVL cloned into the Cla1 site of the pUC μ locus).

b) Introduction of the cosmid into HeLa cells
1 ml of DMEM + 2% TES is fed to HeLa cells (3 · 10 ⁴ cells per dish with a diameter of 6 cm) and they are incubated together with the DNA complexes and transferrin and polylysine conjugate (TfpL) complexes described in FIG. 22A amounts of hTfpL, free polylysine (pL) and DNA. The incubation medium additionally includes either 100 μM chloroquine (see traces 1, 2) or 10 μl of adenovirus d1312 containing 5 • 10 ¹¹ particles per ml (traces 3-12). After two hours of incubation at a temperature of 37 ^o C add 4 ml of DMEM + 10% TES. After 24 hours, cells were harvested to determine luciferase activity. The results of the experiment are presented in FIG. 22A.

c) The introduction of cozmids in neuroblastoma cells
1 ml of DMEM + 2% TES is fed to the cells of the neuroblastoma cell line Gl-ME-N (1 x 10 ⁶ cells per dish with a diameter of 6 cm), after which they are incubated together with the DNA and TfpL conjugate complexes shown in Fig. 22B amount of hTfpL, free polylysine pL and DNA. In addition, the incubation medium additionally contains either 100 μM chloroquine (traces 3, 4) or 10 μl of adenovirus d1312 containing 5 • 10 ¹¹ particles per ml (traces 5, 6). After two hours of incubation at a temperature of 37 ^o C add 4 ml of DMEM medium containing 10% TES. After 24 hours, cells are harvested to determine luciferase activity. The experimental results are presented in Fig. 22B.

Example 17. Gene transfer using a conjugate of chemically related adenovirus and polylysine
a) Obtaining a conjugate of adenovirus and polylysine by chemical binding.

2.35 ml gel-filtered (using a Sephadex G-25 column) adenovirus d1312 solution (approximately 10 ¹¹ particles) in 150 mM sodium chloride / 25 mM HEPES, pH 7.9 + 10% glycerol is mixed with 10 μl (10 nmol) 1 mM solution of succinimidyl pyridyl dithiopropionate. After 3.5 hours at room temperature, the modified virus is separated from the excess reagent by gel filtration, as described above. Argon is passed through 2.5 ml of the solution and, under conditions of exclusion of oxygen in an argon atmosphere, it is reacted with 42 μl of a solution of 1 nmol of fluorescein-labeled polylysine labeled with isothiocyanate, modified with 2.3 nmol of mercaptopropionate groups. After 18 hours at room temperature, half of the solution was transferred to a test tube, 1 ml of a solution of cesium chloride (density 1.33 g / ml) was fed to it and centrifuged at 35,000 rpm for two hours at room temperature. The virus is collected as 200 μl of the cesium chloride fraction and diluted to 1 ml with HBS buffer containing 50% glycerol. 300 μl of the modified virus solution was tested for DNA binding ability as follows. The virus solution is diluted with 1 ml of HBS buffer and mixed with 100 μl of a solution of 15 ng pRSVL DNA labeled with ³⁵ S. As a control experiment, unmodified d1312 virus was used in the same amount. After 30 minutes, the samples are transferred to test tubes, 1 ml of a solution of cesium chloride (density 1.33 / ml) is fed to them and centrifuged at 35,000 rpm for two hours at room temperature. The gradient is divided into 5 fractions: fraction 1 = 1 ml; fraction 2 = 0.6 ml; fractions 3-5 = 200 μl each. The radioactivity of the 200 ml fraction was determined. The data obtained are presented in FIG. 23. A virus containing fractions 3-5, in particular fraction 3, exhibits significantly higher radioactivity than a control sample. This can be attributed to the specific association of polylysine-modified adenovirus with labeled DNA, and in the presence of cesium chloride, it may cause partial dissociation of the complex.

b) Transfection of K562 cells
K562 cells (ATCC CCL 243) are grown in suspension in RPMI 1640 medium (containing 2 g / l sodium bicarbonate, 10% TES, 100 units / ml penicillin, 100 μl / μl streptomycin and 2 mm glutamine) to a density of 500,000 cells / ml . 12 to 20 hours before transfection, the cells are fed into fresh medium containing 50 μM desferrioxime (which serves to increase the number of transferrin receptors). On the day of transfection, cells are harvested, suspended in fresh medium containing 10% TES and 50 μM desferrioxime (250,000 cells / ml) and 2 ml portions are served in a 24-well cup. 6, 0.6 and 0.06 μg, respectively, pCMVL DNA in 100 μl of HBS was mixed with 50 μl of polylysine-modified adenovirus (Fig. 24: "pLadeno") or with the corresponding amount (37 μl) of control adenovirus d1312. After 20 minutes, add 12, 1.2 and 0.12 μg, respectively, of the conjugate TfpL190B in 150 μl of HBS. After a further 20 minutes, the mixture was added to K562 cells, which were incubated at 37 ^° C for 24 hours, after which they were collected to determine the luciferase activity as described in the previous examples. In FIG. 24 shows the total luciferase activity of transfected cells.

c) Transfection of HeLa cells
One of the methods for studying the activity of the conjugate of the virus and polylysine is to determine the ability of the conjugate to transfer minimal amounts of DNA (less than 0.1 μg). An increased ability to transfer DNA is expected if the adenovirus is directly linked to the condensed polylysine DNA, since the factors that promote the uptake of the cell (transferrin and adenovirus (or its protein) are directly linked to the transferred DNA. To verify this assumption proceed as follows. the conjugate of polylysine and adenovirus (2.5 μl, approximately 5 • 10 ⁷ viral particles) is transferred to the complex with different amounts (3 μg - 0.0003 μg) of the reporter plasmid in 475 μl of HBS. After 15 minutes After incubation at room temperature, the transferrin and polylysine conjugate is added to each sample in an amount corresponding to the DNA mass (this amount of TfpL conjugate provides full “packaging” (electroneutrality) of 50% plasmid DNA and at the same time provides additional binding site for the virus and polylysine conjugate. After adding the conjugate TfpL mixtures were incubated for 15 minutes, after which each mixture was fed into a 6 cm diameter dish containing 300,000 HeLa cells in 1 ml of DMEM / 2% TES. Cells were incubated at 37 ^° C. for 90 minutes, after which 4 ml of DMEM / 10% TES medium was added. In parallel experiments, an equivalent amount of DNA is transferred to a complex with a twofold mass excess of TfpL conjugate (this amount provides complete DNA condensation) and is used to carry out gene transfer in HeLa cells (in one case in the presence of 25 ml of d1312 adenovirus unbound with polylysine, in the other case without the last). After 24 hours, cells are harvested, extracts are prepared, and aliquots are examined for luciferase activity. The results of the experiments are presented in FIG. 25, where it can be seen that in the absence of adenovirus, luciferase activity cannot be determined in an amount of DNA of less than 0.3 μg. Both adenovirus bound to polylysine and adenovirus unrelated to it exhibit good effect in the case of large amounts of DNA (3 μg and 0.3 μg). However, in the case of adenovirus unrelated to polylysine, an approximately 100-fold decrease in activity is observed at 0.03 μg of DNA, and in the case of using less than 0.03 μg of DNA, the activity is negligible. In contrast, polylysine-bound adenovirus retains its gene transfer capacity at both 0.003 and 0.0003 μg of DNA. This amount of DNA corresponds to about 150 DNA molecules per cell and about one viral particle per cell.

Example 18. Gene transfer using enzymatically linked to polylysine adenoviruses
a) Reaction with the enzyme
2 ml of an adenovirus preparation (strain d1312; 5 • 10 ¹⁰ plaque forming units / ml) is subjected to gel filtration in a Sephadex G-25 column, which is preliminarily equilibrated with 25 ml of reaction buffer (0.1 M Tris-HCl; pH 8.0 , 2 mM dithiothreitol, 30% glycerol). Elution is carried out with 3.5 ml of reaction buffer. The enzymatic binding reaction mixture consists of 1150 μl of virus-containing eluate, 0.5 nmol of transglutaminase (hereinafter: “TG”) isolated from liver of guinea pig, 2 nmol or 20 nmol of polylysine with a molecular weight of 290, 10 mm calcium chloride and the reaction buffer to the final volume of 1500 μl. The reaction is carried out at a temperature of 37 ^o C for 1 hour, after which it is stopped by adding 30 μl of 0.5 M EDTA. In order to determine the binding specificity, the same reaction mixtures were prepared, but without transglutaminase. Unbound polylysine is separated from viruses by centrifugation in a gradient of cesium chloride (density 1.33 g / ml; 170,000 • g, 2 hours). The virus-containing fraction is collected, mixed with the same volume of glycerol, frozen in liquid nitrogen and stored at a temperature of -70 ^o C.

b) Analysis of the binding of polylysine to adenovirus
The above reaction is carried out using polylysine labeled with ¹²⁵ J. After centrifugation in a gradient of cesium chloride, the virus-containing fraction is withdrawn and separated using another gradient of cesium chloride. The gradient is fractionated and radioactivity is determined in each fraction using a scintillation counter. The results of the experiment are presented in FIG. 26, where it can be seen that in the case of a TG-containing reaction mixture (d1312 / TG-pL), radioactive polylysine accumulates in the virus-containing fraction. In the case of the control mixture, i.e., the reaction mixture without the content of TG (d1312 / pL), the accumulation of radioactive polylysine in the virus-containing fraction is not observed.

c) Investigation of fractions containing polylysine-modified adenovirus, their effect on transfection efficiency
1) Cells and medium
5 • 10 ⁵ cells (murine hepatocytes; ATCC N: TIB 73) in DMEM medium containing 10% inactivated heat treatment of TPP, 2 mM glutamine, 100 units / ml penicillin and 100 μg / ml streptomycin are grown in 6 cm diameter dishes.

2) Formation of a complex consisting of virus, DNA and transferrin
50 μl of the modified polylysine virus-containing fraction was mixed with 6 μg of plasmid DNA pCMVL in 10 μl of HBS and the mixture was incubated at room temperature for 20 minutes. Then, 8 μg conjugate of mouse transferrin and polylysine with a molecular weight of 290 (mTflpL 290B) was added to the mixture, after which the incubation was continued for a further 20 minutes.

3) Transfection of murine hepatocytes
The complex consisting of a modified virus, DNA and transferrin is mixed with 1.5 ml of DMEM medium containing 2% TES, 2 mM glutamine and antibiotics, and added to the cells after preliminary replacement with fresh medium. After two hours of incubation at 37 ^° C, 2 ml of DMEM medium containing 10% TES, glutamine and antibiotics are added to the cells. After cultivation for a further 2 hours, the whole medium is removed and 4 ml of fresh DMEM medium containing 10% TES, glutamine and antibiotics are added to the cells.

4) Determination of luciferase expression
24 hours after transfection, cells are harvested and luciferase activity determined in the manner described above. As seen in FIG. The 27th viral preparation containing the treated TG and 20 nmol of polylysine (d1312 / TG-20 nmol pL) adenovirus shows the highest expression (153,540,000 light units). A viral preparation containing adenovirus treated with TG and 2 nmol of polylysine (d1312 / TG-2 nmol pL) is slightly less active (57,880,000 light units). The control fraction, which contains only processed 20 nmol of polylysine of adenovirus, exhibits approximately a factor of 500 less activity. As a comparison, complexes are used that contain an adenovirus that is not treated with either TG or polylysine. The luciferase activity is 4,403,000 light units.

5) The efficiency of transfection achieved using polylysine-modified adenovirus compared to unmodified adenovirus can be increased in particular by small amounts of DNA
Transfection is carried out as described in step 3) using 50 μl of the adenovirus fraction d1312 / TG-20 nmol pL and 6 μg pCMVL / 8 μg TfpL, 0.6 μg pCMVL / 0.8 μg mTfpL or 0.06 μg pCMVL / 0. 08 μg mTfpL to form a complex. As a comparison, transfection was performed using 6 μg, 0.6 μg and 0.06 μg, respectively, of the pCMVL / mTfpL complex and unmodified adenovirus d1312. The results of the experiment are presented in FIG. 28. It is seen that the complexes containing the polylysine-modified adenovirus exhibit high expression even in the case of small amounts of DNA, while the expression decreases sharply when using a complex containing unmodified adenovirus.

Example 19. Gene transfer using conjugates in which the connection between adenovirus and polylysine is carried out using a biotin-streptavidin bridge
a) Biotinylation of adenovirus d1312
2.4 ml gel-filtered in a Sephadex G-25 column of adenovirus d1312 solution (approximately 10 ¹¹ particles) in 150 mM sodium chloride / 5 mM HEPES, pH 7.9 / 10% glycerol is mixed with 10 μl (10 nmol) 1 mm solution of biotin brand NHS-LC. After 3 hours at room temperature, the biotin-modified virus is separated from the excess reagent by gel filtration, as described above. Glycerin is added to the solution in an amount that ensures its concentration in the solution of 40% and the solution with a total volume of 3.2 ml is stored at -25 ^o C. The fact of biotinylation of the virus is investigated by dropping a solution of varying degrees of dilution onto a cellulose-nitrate membrane, and then drying is carried out at temperature of 80 ^o C for 2 hours in a vacuum dryer, albumin blocking of cattle serum, incubation with streptavidin-associated alkaline phosphatase, washing and one-hour incubation with the developing solution tetrazole salt of nitrosine and toluidine salt of 5-bromo-4-chloro-3-indolylphosphate. At the same time, there is a positive color reaction, which indicates the completed biotinylation.

b) Obtaining conjugates of streptavidin and polylysine
79 nmol (4.7 mg) of streptavidin in 1 ml of 200 mM HEPES buffer with a pH of 7.9 and 300 mM sodium chloride is treated with a 15 mM ethanolic solution of succinimidyl pyridyl dithiopropionate (236 nmol). After 90 minutes at room temperature, the modified protein is subjected to gel filtration in a Sephadex G-25 column. In this case, 75 nmol of streptavidin modified with 196 nmol of the indicated dithiopyridine are obtained. The modified protein is reacted with 75 nmol of the modified 190 nmol of polylysine 3-mercaptopropionate with an average length of lysine monomers of 290 in 2.6 ml of 100 mM HEPES buffer with a pH of 7.9 containing 150 mM sodium chloride. Conjugates were isolated by cation exchange chromatography on a Mono S HR5 column (gradient: 20-100% buffer consisting of 50 mM HEPES pH 7.9 plus 3 M sodium chloride). The target fraction is eluted at a salt concentration between 1.2 M and 1.7 M. As a result of dialysis using HBS buffer (20 mM HEPES with pH 7.3, 150 mM sodium chloride), a conjugate of 45 nmol streptavidin and 53 nmol are obtained polylysine.

c) Transfection of HeLa cells
HeLa cells are grown in 6 cm diameter dishes as described in Example 13. Transfection is carried out at a density of 300,000 cells per dish. Before transfection, cells are incubated in 1 ml of fresh medium containing 2% TES.

 6 μg of pCMVL DNA in 100 μl of HBS was mixed with 0.8 μg of streptavidin-modified polylysine in 170 μl of HBS. After 20 minutes, add 3 μg of polylysine with a molecular weight of 300 in 170 μl of HBS. After a further 20 minutes, add 65 μl of biotinylated adenovirus or, as a control, the corresponding amount of adenovirus d1312 (30 μl, i.e. the amount in which the virus is used to modify). The resulting mixture ("Biotin.AdV / complex A" and "AdV (control)"; see Fig. 29) is left to stand for a further 20 minutes.

 In addition, 50 μl of biotinylated adenovirus is mixed with 0.8 μg of streptavidin-modified polylysine in 50 μl of HBS, 6 μg of pCMVL DNA in 170 μl of HBS is added after 20 minutes and another 3 μg of polylysine with a molecular weight of 300 to 200 μl is added after a further 20 minutes Hbs. The resulting complex is designated "BiotinAdV / Complex B".

 0.6 μg of pCMVL DNA in 67 μl of HBS was mixed with 0.3 μg of streptavidin-modified polylysine in 33 μl of HBS. After 20 minutes, add 65 μl of biotinylated adenovirus or, as a control, an appropriate amount of adenovirus d1312 (30 μl, i.e. the amount in which the virus is used to modify). The resulting complexes (“biotin. AdV / complex A” or “AdV (control)”; see FIG. 29) were left to stand for a further 20 minutes, after which the complexes were diluted with HBS to 500 μl. In addition, another complex is obtained by mixing 50 μl of biotinylated adenovirus with 0.3 μg streptavidin-modified polylysine in 50 μl HBS and then adding 0.6 μg pCMVL DNA in 50 μl HBS after 20 minutes. The resulting complex (Biotin.AdV / Complex B) was left to stand for a further 20 minutes, after which it was diluted with HBS to 500 μl.

These complexes are added to cells, which are incubated at a temperature of 37 ^o C for 2 hours, after which 2.5 μl of fresh medium containing 10% TES. Cells were incubated for an additional 24 hours at 37 ^° C, after which they were collected to determine luciferase activity as described above. The experimental results are presented in Fig. 29, showing the total luciferase activity of transfected cells.

 At the same time, HeLa cells are transfected using the biotinylated virus conjugate inactivated by treatment with UV rays and psoralen as the viral component. Inactivation is as follows. Samples of 200 μl of biotinylated virus are fed into a 1.6 cm diameter two-well plate. 2 μl (33 mg / ml) of 8-methoxy psoralen in dimethyl sulfoxide are added to each sample, the plates are fed onto ice and irradiated with UV light using a UVP lamp TL-33, placed at a distance of 4 cm, for 10 minutes at 365 nm. After irradiation, both samples are combined and subjected to gel filtration in a G50 column, which is pre-equilibrated with a 40% solution of glycerol in HBS. Aliquots of 75 μl were transferred to a complex with 0.8 μg of streptavidin-modified polylysine and the complexes thus obtained were used to transfect HeLa cells as described above.

As a result of cytopathic analysis, it is established that inactivation leads to a decrease in the titer of the virus by a factor of more than 10 ⁴ , while the ability to transfer the gene decreases by less than 50% at high concentrations and by factor 5 at low concentrations.

g) Transfection of K562 cells
K562 cells in suspension are grown in RPMI 1640 medium containing 2 g / l sodium bicarbonate, 10% TES, 100 units / ml penicillin, 100 μg / ml streptomycin and 2 mM glutamine, until a density of 500,000 cells / ml is reached. 16 hours before transfection, the cells are fed into fresh medium containing 50 μmol of desferrioxime. On the morning of transfection, the cells are harvested, resuspended in fresh medium containing 10% TES (plus 50 μM desferrioxime) at a density of 250,000 cells / ml and served in a 24-well cup (2 ml per well).

3 different DNA complexes are prepared: a) A solution of 6 μg pCMVL DNA in 160 μl HBS (150 mM sodium chloride, 20 mm HEPES with pH 7.3) is mixed with 12 μg TfpL190B conjugate in 160 μl HBS, 20 μl of adenovirus is added after 30 minutes d1312 and the resulting mixture was added to K562 cells. b) A solution of 800 ng of streptavidin-modified polylysine (StpL) in 160 μl of HBS is mixed with 20 μl of the biotinylated adenovirus obtained in the above step a), after 30 minutes a solution of 6 μg of pCMVL DNA in 160 μl of HBS is added, and after a further 30 minutes the solution is mixed with 10 μg TfpL190B conjugate in 160 μl HBS. After 30 minutes, the mixture is added to the cells. c) This complex is prepared analogously to point b) with the difference that instead of the TfpL190B conjugate, a solution of 3.5 μg poly (L) lysine with a molecular weight of 290 (pL290) is added. Cells are incubated at a temperature of 37 ^o C for 24 hours, after which they are collected to determine the activity of luciferase. The results of the experiments are presented in FIG. 30, showing the total luciferase activity of transfected cells.

Example 20. Gene transfer to primary bone marrow cells
a) Isolation of primary bone marrow cells
Primary bone marrow cells were selected from mice using a 1 ml syringe equipped with an injection needle (0.4-0.5 mm in diameter) inserted into an isolated femur and tibia. The selection is carried out using IMDM medium containing 10% TES, 5 • 10 ^-5 M β-mercaptoethanol, 1% interleukin 3 and antibiotics. Selected cells are washed with the indicated medium by centrifugation at 100 x g for 8 minutes. Then the cells are resuspended in the indicated medium at a concentration of 10 ⁷ / ml and cultured in T25 brand tubes. After 4 hours, the sprouted cells are transferred to other T25 tubes in which they are cultured overnight in the presence of 50 μM desferrioxime.

b) Formation of a complex consisting of adenovirus, DNA and transferrin
50 μl of biotinylated adenovirus are incubated with 400 ng of streptavidin-modified polylysine in 20 μl of HBS for 20 minutes. Then add 20 μl of HBS buffer containing 6 μg of pCMVL DNA. After a 20-minute incubation, add 7 μg of mouse transferrin conjugate and polylysine (mTfpL) in 160 μl of HBS, after which the mixture is incubated for a further 20 minutes.

c) Transfection
Bone marrow cells are separated from the culture medium by centrifugation at 100 x g for 8 minutes. The resulting cells are resuspended in 3 ml of the indicated medium containing 2% TES and 250 μl of the complex consisting of adenovirus, DNA and transferrin, after which the mixture is cultured in T25 brand tubes at a temperature of 37 ^° C for 3 hours. Then add 3 ml of the specified medium containing 10% TPP. After a further 2 hours, another 6 ml of this medium is added.

g) Determination of luciferase expression
After a 48-hour transfection, bone marrow cells are harvested and analyzed for luciferase expression as described above. It was established that transfection leads to the expression of luciferase activity corresponding to 310 • 10 ³ light units / 100 μg of total cellular protein.

Example 21. Transfection of neuroblastoma cells with a cosmid of 48 kp about. in the presence of adenovirus
a) Obtaining a cosmid comprising a sequence encoding luciferase
The 3.0 kbp Sal I fragment, including the luciferase coding sequence from P. pyralis under the control of the RSV promoter, is doped into the only Sal I site of the cosmid clone C1-7aA1 to obtain a concamer (C1-7aA1 includes the Sau 3A fragment 37 kbp of human genomic DNA (partial digestion), not encoding specific genes, cloned into the Bam HI site of the cosmid vector pWE15). The doping product is packaged in vitro and an aliquot of the resulting phage particles is infected with E. coli strain NM544, which is fed onto LB amp plates. The resulting recombinant products were screened by colony hybridization using a 3.0 kbp Sal I fragment labeled with ³² P. as a probe. A number of positive products were subjected to restriction mapping analysis. The cosmid construct (CosLuc), including one single copy of the Sal I insert, is grown and purified on a gradient of cesium chloride (total size: 48 cbp).

 A small control plasmid pWELuc of 12 kbp was obtained. by digesting the specified cosmid construct using Not I, re-doping, transforming bacteria and isolating a clone including the target plasmid. A 12 kbp DNA molecule is obtained in which there is no human DNA insert and part of the CosLuc cosmid construct polylinker.

b) The introduction of cosmids in neuroblastoma cells
A layer of 1 ml of DMEM medium containing 2% TES is fed onto the cells of the neuroblastoma line Gl-ME-N (1 x 10 ⁶ cells per dish with a diameter of 6 cm), after which the cells are incubated together with the above DNA complexes and TfpL complexes containing the indicated amounts hTfpL, free polylysine and DNA. The incubated mixtures also contain either 100 μM chloroquine (see traces 3, 4) or 10 μl of adenovirus d1312, including 5 • 10 ¹¹ particles / ml (see traces 5, 6). Both last samples (designated as StpL / Biotin.), Including 15 μl of biotinylated adenovirus d1312 (1 x 10 ¹¹ particles), were incubated with 0.8 μg of the streptavidin-modified polylysine modified in 150 μl HBS for 30 minutes. Then add 6 μg of DNA in 150 μl of HBS, incubate at room temperature for 30 minutes and add 150 μl of HBS buffer containing 6 μg of hTfpL and 1 μg of free polylysine. After an additional incubation at room temperature for 30 minutes, the mixture was added to the cells, which were incubated at 37 ^° C for 2 hours, after which 4 ml of DMEM medium containing 10% TES was added to each sample. After 24 hours, cells were harvested to determine luciferase activity. The results of the experiment are presented in FIG. 31.

Example 22. Gene transfer to primary epithelial airway cells using the conjugates of the invention. Studies aimed at identifying the suitability of using gene transfer using the conjugates of the invention for the genetic correction of cystic fibrosis have shown that immortalized cell lines from human airway epithelium are sensitive to this method of gene transfer. To exclude the fact that this defect is the result of immortalization-induced changes in the airway epithelium, primary airway epithelial cells (hereinafter: 1 ^o AE cells) are exposed to conjugates of transferrin and polylysine. Cells 1 ^o AE isolated from the nasal polyp of a person by known methods. The tissue is subsequently washed with sterile saline and MEM medium containing 50 units / ml penicillin, 50 μg / ml streptomycin and 40 μg / ml gentamicin and transferred to the laboratory at 4 ^o C. The cartilage and excess submucosal tissue are cleaned and the epithelial layers are incubated in containing a protease (type 14 company Sigma, DE; 0.1 mg / dl) solution in MEM at a temperature of 4 ^o C for 16 to 48 hours. 10% TES is added to neutralize the protease, after which the mixture is slightly stirred. The resulting suspension is filtered through a nylon mesh with a mesh size of 10 μm in order to remove foreign substances, and the filtrate is subjected to centrifugation at 150 g for 5 minutes, after which it is washed in F12 medium containing 10% TES. The 1 ^o AE cells are then treated with conjugates of transferrin and polylysine (hTfpL) containing the luciferase encoding plasmid (pRSVL) as a reporter gene. In this experiment, primary cells do not show the sensitivity to the action of this vector, which is manifested by the corresponding immortalized cell lines (1 ^o AE: background = 429 ± 41; + hTfpL = 543 ± units of luciferase), which probably indicates a relatively small number transferrin receptors on cells 1 ^o AE.

In order to study an alternative target receptor on 1 ^o AE cells, conjugates are constructed that include an adenovirus (bAdpL; replication incompetent as a ligand; see Example 19 a) and 19 b) to obtain biotinylated adenovirus and streptavidin and polylysine conjugates; a luciferase encoding plasmid is used as a reporter gene). Human 1 ^o AE cells treated with this conjugate exhibit a reporter gene expression level that significantly exceeds background (+ bAdpL = 2 585 753 ± 453 585 luciferase units). In addition, 1 ^o AE cells of other origin also exhibit a high degree of sensitivity to gene transfer by this method (from mice = 3 230 244 ± 343 153; from monkeys = 53 498 880 ± 869 481 units of luciferase). Thus, the ability to carry out gene transfer into 1 ^o AE cells confirms the potential suitability of this approach for direct gene delivery in vivo.

Example 23. Gene transfer to hepatocytes using the proposed conjugates
a) Transfection of cells
Cells of the mouse embryonic hepatocytic cell line BNL CL.2 (ATCC TIB 73) are grown as described in example 6. HeLa cells and hepatocytes are grown in plastic Petri dishes with a diameter of 6 cm. Transfection is carried out at a cell density of about 3 • 10 ⁵ / cup. Before transfection, the standard nutrient medium is replaced with 1 ml of fresh medium containing 2% TPP.

b) The formation of a double complex
Biotinylated adenovirus (approximately 10 ⁹ plaque forming units; see Example 19 a) and 19 b)) is reacted with 800 ng of streptavidin-modified polylysine in 50 μl of HBS. After 30 minutes at room temperature, add 6 μg of pCMVL DNA in 170 μl of HBS, incubate for 30 minutes and add 3 μg of polylysine with a molecular weight of 300 in 200 μl of HBS. After a further 30 minutes, the solution is used to effect transfection.

c) Formation of a triple complex
A triple DNA complex containing adenovirus and transferrin is prepared as follows. Biotinylated adenovirus (approximately 10 ⁹ viral particles) is mixed with 800 ng of streptavidin-modified polylysine. After 30 minutes at room temperature, the solution was mixed with 6 μg of plasmid DNA in 170 ml of HBS, incubated for 30 minutes, after which 10 μg of polylysine conjugate and TfpL 190B transferrin in 200 μl of HBS were added. After a further 30 minutes, the solution is used to effect transfection.

g) Determination of β-galactosidase
Cells BNL CL. 2 are seeded on coverslips and after 24 hours the cells are transfected with the pCMV-β-gal reporter gene. After 48 hours, the content of β-galactosidase is determined.

 Double complexes used for transfer. Biotinylated adenovirus is combined with modified streptavidin polylysine. Alternatively, adenovirus is covalently bound to polylysine via transglutaminase. The conjugate of adenovirus and polylysine is added to the DNA under conditions ensuring the formation of a complex between DNA and polylysine. In this case, a certain part (approximately 1/4) of the negative charges of DNA is neutralized. Then, the calculated amount of polylysine is added in order to neutralize the remaining negative charges. DNA complexes associated with the conjugate of adenovirus and polylysine and with polylysine are hereinafter referred to as double transfer complexes.

 Basically, there are two ways to obtain double transfer complexes. DNA can first be bound to streptavidinylated polylysine and then to biotinylated adenovirus, or the adenovirus is first bound to polylysine and then converted to a DNA complex. The latter method gives much better results, in particular at low DNA concentrations. Therefore, this approach is the preferred method for producing binary and triple complexes.

 Triple transfer complexes containing transferrin. The conjugates of adenovirus and polylysine are added to the DNA under conditions that ensure the formation of the complex and the neutralization of part (about 1/4) of the negative charges of DNA. Then, the calculated amount of the transferrin conjugate and polylysine is added to the complex in order to neutralize the remaining negative DNA charges. Complexes consisting of DNA, conjugates of adenovirus and polylysine and transferrin and polylysine are hereinafter referred to as triple complexes. In principle, such a triple complex should be capable of being incorporated by the cell as a result of binding to adenovirus cell receptors or transferrin receptors.

The relationship between DNA condensates and adenovirus significantly improves the expression of the luciferase reporter gene. The physical connection between the adenovirus strain d1312 and polylysine can be achieved either by incubation of both components together with transglutaminase, or by biotinylation of adenovirus and streptavidinylation of polylysine. The effect of such binding on transfection efficiency is clearly shown in FIG. 32, which presents the results of incubation of hepatocytes together with DNA and conjugate complexes of transferrin and polylysine (TfpL) in the presence of chloroquine or in the presence of adenovirus (AdV + TfpL). Transferrin infection, i.e. Transferin-facilitated transfection, in the presence of free adenovirus, is improved so that it leads to a typical increase in the release of DNA complexes and the conjugate of transferrin and polylysine into cells. In the case of pLAdV / TpfL complexes, adenovirus is conjugated to polylysine using transglutaminase and then reacted with DNA, which leads to the neutralization of some of the negative charges of DNA. Then the conjugate of transferrin and polylysine is added to neutralize the remainder of the negative charges. In this way, a triple complex of DNA and conjugates of adenovirus and polylysine and transferrin and polylysine is synthesized. It can be seen that in this case, an extremely high degree of luciferase expression can be achieved, i.e. 1.5 • 10 ⁹ light units of luciferase (approximately 5000 light units per cell). In the case of AdV + pL + TfpL complexes, the adenovirus is mixed with polylysine in the same manner as in the case of transglutaminase treatment. In order to determine the specificity of the transglutaminase-facilitated binding of polylysine to the virus, the process is carried out in the absence of an enzyme. Then the virus preparation is transferred to the complex with the same amount of DNA and TfpL as in the pLAdV / TfpL complexes. In this case, transfection is moderate as in the case of AdV and TfpL-containing complexes, since in both experiments the mutual placement of the virus, transferrin and DNA is a random process, whereas in the case of pLAdV and TfpL-containing complexes, the inclusion of the cell is provided by physical binding of the virus with DNA in a triple complex. At the same time, a high degree of transferrin infection is achieved.

 Transfection of K562 cells shows the endosomolytic properties of adenovirus. The K562 human erythroleukemia cell line contains approximately 150,000 transferrin receptors. As mentioned above, in the presence of chloroquine, these cells can be subjected to highly efficient transferrin infection by treatment with complexes of reporter DNA and conjugate of polylysine and transferrin even in the absence of adenovirus (TfpL, Fig. 33). The same complexes with added free adenovirus (AdV / TfpL), but in the absence of chloroquine, provide only a relatively small degree of expression of the reporter gene, presumably due to the fact that K562 cells, like other blood cells, have a low number of adenovirus receptors . If adenovirus is bound to polylysine via a biotin-streptavidin bridge and the reporter DNA is completely condensed by adding the appropriate amount of polylysine to obtain the triple complex (pLAdV / pL), the degree of adenovirus-supported transfection is moderate. It is believed that a small number of adenovirus receptors on K562 cells are used in an efficient manner. However, if the reporter DNA associated with adenovirus and polylysine is completely condensed and neutralized by the addition of a transferrin conjugate and polylysine to obtain the pLAdV / TfpL triple complex and there are numerous cellular transferrin receptors, then, thanks to the effective binding of the transferrin and the endosomolytic properties of the virus, the transfection efficiency increases by at least 2 orders of magnitude (see Fig. 33).

 Triple DNA complexes provide expression of the reporter gene in almost all hepatocytes. The effectiveness of the proposed DNA complexes is also investigated on murine hepatocytes (BNL CL.2) by determining the percentage of their transfection. Moreover, the β-galactosidase gene under the control of the CMV promoter is used as the reporter gene. After cell fixation, β-galactosidase activity is determined.

 In FIG. 34 presents the results of an analysis of β-galactosidase activity in the case of murine hepatocytes after A) a transferrin infection in the presence of chloroquine, B) a transferrin infection in the presence of free adenovirus d1312 and C) transfection with a triple complex consisting of adenovirus d1312, polylysine, transferrin and reporter DNA. After the usual transferrin infection of the reporter DNA in the absence of adenovirus, only a few cells express the reporter gene. Percentage transfection is less than 0.1%. If chloroquine is added, the percentage of transfection rises to about 0.2% (see FIG. 34A). In the presence of free adenovirus, 5-10% of the cells express the reporter gene (see FIG. 34B), while ternary complexes including transglutaminase-modified viruses provide expression in almost all cells (see FIG. 34C). Since the ternary complex can be used in a highly diluted form, the toxic effect that is observed in the case of high doses of free (inactivated) adenovirus usually does not occur. But it should be noted that in the case of the use of the triple complex in a high concentration, which ensures the involvement of 100% of the cells in the process, a similar toxic effect begins to appear. Toxic effects may result from the residual activity of the viral gene, the endosomolytic properties of the added virus, or they may simply cause a very high level of expression of the transfected gene.

 Expression of the transfected reporter gene is temporary, but continues for weeks in non-fissile hepatocytes. Ternary complexes (pLAdV / TfpL) are prepared, including the polylysine-modified adenovirus d1312 and the modified adenovirus d1312 inactivated by treatment with psoralen. Similar to the experience of FIG. 34B 2/3 of the fused hepatocyte cell culture is transfected with the CMVL luciferase reporter gene and luciferase activity is determined at different times. As seen in FIG. 35, luciferase activity is maximum after three days, that is, at the time of fusion of the cell culture and cessation of cell division. Expression of the reporter gene continues in non-dividing cells for at least 6 weeks without the need for selection to ensure gene viability, in particular when an adenovirus inactivated by psoralen treatment is used to form a triple complex.

 Example 24. The use of chicken adenovirus CELO to improve the transfer of DNA into human cells.

2 ml of chicken CELO adenovirus (Phelps strain, serotype FAV-1.7) was subjected to gel filtration in a PD-10 column, which was pre-equilibrated with 20 mM HEPES pH 7.3 buffer and 150 mM sodium chloride buffer containing 10% glycerol. 2 ml of the resulting eluate is reacted with 20 μl of 1 mM NHS-LC biotin at room temperature for 3 hours. The resulting biotinylated virus is dialyzed using 3 x 300 ml HBS buffer containing 40% glycerol at a temperature of 4 ^o C, after which aliquots of the virus are stored at a temperature of -70 ^o C. HeLa cells (5 • 10 ⁵ cells per cup with a diameter of 6 cm ) in 2 ml of DMEM medium containing 2% TES, incubated with 6 μg of plasmid pCMVL, available as a complex with polylysine (pL) or conjugate of transferrin and polylysine (TfpL) in 500 μl of HBS buffer (the complex is preincubated at room temperature for 30 minutes). Then the samples are added to the cells at a temperature of 37 ^o C in the presence of the virus, taken in the amount indicated in Fig. 36. In the case of samples containing the biotinylated CELO virus, the indicated amount of virus is pre-incubated with the indicated amount of streptavidin-modified polylysine (StpL) in 200 μl of HBS buffer at room temperature for 30 minutes, after which 6 μg of pCLus plasmid in 100 μl of HBS is added . After a 30-minute incubation at room temperature, the indicated amount of the TfpL-containing complex is added to the cells at a temperature of 37 ^° C. After 2 hours, 5 ml of DMEM medium containing 10% TES is added to the cells, and after 24 hours the cells are collected and submitted for activity determination luciferase. The results of the experiment are presented in FIG. 36.

 As seen in FIG. 36, the CELO virus in its free form improves the delivery of DNA to HeLa cells (see tracks 1 to 6). However, if the CELO virus is modified with biotin and complexed together with streptavidin, either with the transferrin and polylysine conjugate, or without it, the virus provides an improvement in DNA delivery to a degree that can be compared with the case when human adenovirus d1312 is used. This HeLa cell line has a large binding capacity comprising polylysine and DNA complexes in the absence of transferrin (cf. luciferase activity of samples 1 and 4 in Fig. 36). Thus, the inclusion of the CELO virus in the complex of polylysine and DNA is sufficient to "start" the absorption of the virus.

Example 25. Transfection of myoblasts
a) Transfection of myoblasts and myotubes with DNA, transferrin and polylysine complexes in the presence of free adenovirus and in the presence of biotinylated and streptavidinylated adenovirus.

C ₂ Cl ₂ myoblasts (N ATCC: CRL 1772) and G8 myoblasts (ATCC No: CRL 1456) are grown in DMEM containing high glucose concentration containing 10% TES. Myoblast cultures are transfected with approximately 5 x 10 ⁵ cells per dish 6 cm in diameter. Myotubule cultures are obtained by feeding myoblasts into 6 cm dia cups (approximately 5 x ^{10 5} cells per dish) and replacing the medium with a high glucose concentration DMEM, containing 2% horse serum when cell fusion is achieved. Transfection of myotubules is carried out after 5 to 7 days. The complexes used for transfection are prepared in the manner described in Example 19 using those indicated in FIG. 37 amounts of TfpL, StpL and biotinylated adenovirus d1312. 20 hours after transfection, cells were harvested to determine luciferase activity. The results of the experiment are presented in FIG. 37, where luciferase activity for the entire sample is indicated in light units. It is seen that both myoblasts and myotubules can be transfected with high efficiency. After differentiation into myotubules, a decrease in transfection efficiency by less than 1 log is observed (in the case of C ₂ Cl ₂ ) or no significant decrease is observed (in the case of G8). In the case of the G8 line, participation in the formation of myotubules takes place with a lower frequency, for which the absence of a definite decrease in efficiency in a differentiated culture may be partially responsible. In the case of this cell, the interaction between transferrin and its receptor in DNA transfer is not critical. In all 4 cell preparations, only weak DNA delivery is observed when using TfpL and DNA complexes in the presence of free adenovirus d1312 (see traces 1, 4, 7, 10). Transfection efficiency increases in the presence of biotinylated streptavidin-modified adenovirus (see traces 2, 3, 5, 6, 8, 9, 11, 12). The delivery efficiency achieved by using a combination complex that includes only the virus, pL and StpL increases by less than 1 log compared to complexes that include only the transferrin and polylysine conjugate (TfpL) (compare, for example, trace 2 (without transferrin) with a trace of 3 (in the presence of transferrin)). Regarding insignificant transfection in the case of the use of the free virus and high transfection in the case of the use of complexes containing the bound virus, either in the presence or in the absence of the conjugate of transferrin and polylysine, it is assumed that adenovirus serves as a ligand in these cells, and in the absence of binding, the free virus may penetrate into the cells, however, the complex of DNA and the conjugate of transferrin and polylysine does not penetrate efficiently (in this example, pCMVL is used as DNA).

b) Histochemical analysis of the frequency of transfection in myotubules
Similarly to stage a), cultures of C ₂ Cl ₂ myotubules are prepared (5 × 10 ⁵ cells are seeded into 6 cm diameter dishes as myoblasts, which are then differentiated into myotubules). 6 μg pCMV β-gal DNA was complexed with 8 μg TfpL in 500 μl HBS buffer and added to the cells in the presence of 18 μl d1312 adenovirus (1 × 10 ¹² viruses per ml) in 2 ml DMEM medium containing 2% TES. In addition, 6 μg of pCMVLacZ DNA was complexed with 7 μg of TfpL and 800 ng of StpL, including 18 μl of biotinylated adenovirus d1312 (1 x 10 ¹² viruses per ml) in 500 μl of HBS buffer and added to cells in 2 ml of DMEM containing 2% TPP. After a 24-hour incubation, cells are stained as described in Example 15 in order to determine β-galactosidase activity. The results of stained samples are consistent with the results of transfection using luciferase as a reporter gene (see paragraph a). In the case of the use of free virus, only very low gene expression is observed in myotube cultures, while the binding of the virus to DNA leads to a high level of gene expression. The presence of blue-stained tubules indicates successful gene transfer to these differentiated cells in the presence of free adenovirus.

c) DNA transfer to primary murine myoblasts and myotubules
The main skeletal muscles of both hind legs of a C57B1 / 6 male male at 4 weeks of age are sterilely isolated in PBS and cut into pieces of approximately 5 mm. Tissue was suspended in 20 ml of PBS, allowed to stand for about 2 minutes and the supernatant was aspirated. These operations are repeated 3 times. The tissue is then mixed with 3.5 ml of PBS buffer containing 0.5 ml of trypsin / EDTA, 0.5 ml of 1% by weight collagenase and 0.5 ml of 1% ACC (fraction V, in 4 mM calcium chloride ) and the mixture is incubated at a temperature of 37 ^o C for 30 minutes with frequent slight stirring. After a 30-minute incubation, the remaining tissues are allowed to settle, the supernatant is removed and mixed with 5 ml of DMEM medium, including 20% TES. The incubation with the protease is repeated 3-4 times until the tissue is completely dispersed. The resulting cell suspension is filtered and centrifuged at 500 g for 15 minutes. The centrifugation product is resuspended in 10 ml of DMEM containing 20% TES and the fibroblasts are removed by plating the cells on uncoated plates with a diameter of 15 cm for 60 minutes. Unbound cells are carefully removed and plated on 5 laminin-coated Petri dishes with a diameter of 10 cm, each of which contains 15 ml of DMEM containing 20% TES. After fusion is achieved (after about one week), the cells are trypsinized and plated again on laminin-coated Petri dishes with a diameter of 6 cm in an amount of about 1 × 10 ⁶ cells per dish. To obtain myotubule cultures, after about 5 days (when cell fusion is achieved), the medium is replaced with DMEM containing 2% horse serum, and the transfection process is carried out after one week. Myoblast cultures are transfected in plates with a diameter of 6 cm at approximately 80% fusion.

 Laminin coated cups are prepared as follows. 0.025 mg / ml polylysine with a molecular weight of 30,000 - 70,000 in sterile water at room temperature for 30 minutes is served in a cup. Then the plates are washed 3 times with sterile water and air dried. The plates are then coated with laminin, supplied in water in an amount of 8 μg / ml, at room temperature overnight, after which the plates are washed 3 times with sterile water before plating the cells.

 The DNA complexes used for transfection are obtained by diluting the one indicated in FIG. 38 amounts of biotinylated adenovirus d1312 inactivated by treatment with psoralen and ultraviolet rays (obtained as described in Example 19; in Fig .: "inact. AdV") in 150 μl of HBS buffer and adding 1 μg of StpL in 150 μl of HBS buffer, followed by incubation at room temperature for 30 minutes. Then, 100 μl of HBS buffer containing 6 μg pCMVL was added to each sample, followed by incubation at room temperature for 30 minutes. Finally, 7 μg TfpL in 100 μl HBS buffer is added to each sample, incubated at room temperature for 30 minutes and added to cultures of myoblasts and myotubules in 6 cm diameter dishes containing 2 ml of DMEM medium containing 2% TES. After a one-hour incubation, the medium is replaced with 5 ml of DMEM medium containing 20% TES (in the case of myoblasts), or 5 ml of DMEM medium containing 2% horse serum (in the case of myotubules). After 48 hours, cells are harvested to determine luciferase activity. The results of the experiment are presented in FIG. 38.

Example 26. Improving the transfer of CELO virus to myoblasts using pectin as a ligand
a) Comparative analysis of adenovirus d1312 and CELO virus in HeLa cells and myoblasts C ₂ Cl ₂
Samples consisting of either HeLa cells or C ₂ Cl ₂ myoblasts (5 x 10 ⁵ cells per dish with a diameter of 6 cm) are transfected with 6 μg pCMVL DNA as a complex with 1 μg StpL / 7 μg TfpL and 5 μl biotinylated adenovirus d1312 (cm Example 19; 1 • 10 ¹² particles / ml) or 18 μl of biotinylated CELO virus (see Example 24; 0.3 • 10 ¹² particles per ml). After a 20-hour incubation, cells are harvested to determine luciferase activity. The results of the experiment are presented in FIG. 39.

Transfection in HeLa cells can be carried out with comparable efficiency both when using human adenovirus d1312, StpL, TfpL and DNA complexes that penetrate the cells either using the adenovirus receptor, or using the transferrin receptor, or consisting of CELO, StpL, TfpL and DNA complexes that can penetrate cells using the transferrin receptor. DNA transfer to C ₂ Cl ₂ myoblasts can be effectively carried out using adenovirus d1312-containing complexes, while CELO virus-containing complexes are less effective in these cells. The results of experiments in the previous examples indicate that the transferrin receptor plays only a minor role in the transfer of the combination complex to these cells. The adenovirus receptor is believed to be the main entry site. The slight activity of the CELO virus in myoblasts may result from poor binding of both the CELO virus and transferrin to C ₂ Cl ₂ myoblasts.

b) Improving the transfection of C ₂ Cl ₂ myoblasts with the CELO virus when using wheat germ agglutinin as a ligand
Due to the ineffective transfer in the experiment under item a), transferrin is replaced with a new ligand, obtained as follows.

CELO biotinylated virus (3 x 10 ⁹ cells) and 1 μg of wheat germ biotinylated agglutinin agglutinin (hereinafter “APZ") in 150 μl HBS buffer are mixed with a solution of 0.8 μg StpL in 150 μl HBS buffer and the mixture is incubated at room temperature for 30 minutes. 6 μg of pCMVL DNA in 100 μl of HBS buffer is added to a solution of the virus, AH and StpL in 300 μl of HBS, followed by incubation at room temperature for 30 minutes. Then, 7 μg TfpL in 100 μl HBS buffer was added to the mixture, after which it was again incubated at room temperature for 30 minutes. The resulting complexes are added to C ₂ Cl ₂ myoblasts (5 • 10 ⁵ cells per dish with a diameter of 6 cm) in 2 ml of DMEM medium containing 2% TES. After an hour, 5 ml of DMEM medium containing 10% TES was added to the cells, and after 20 hours, cells were collected to determine luciferase activity (in light units). The results of the experiment are presented in FIG. 40. In this FIG. it can be seen that in the absence of the virus, DNA transfer is very insignificant both in the presence of APA and in its absence (see traces 1, 6). Transfer of negligible efficacy is achieved with the use of the bound CELO virus (see trace 2). But a 16-fold increase in transfer efficiency is achieved in the case of inclusion in the complex APZ. An increase in the APA content in the complexes (from 1 μg to 5 μg) leads to a slight decrease in the transfer efficiency (cf. traces 3 and 4), whereas as a result of an increase in the StpL content in the complex (from 1 μg to 2 μg), a slight increase in the transfer efficiency is observed (cf. tracks 3 and 5). Thus, the results of this experiment indicate that APA as a ligand provides an increase in the efficiency of DNA transfer into C ₂ Cl ₂ cells using the CELO virus.

d) Expression of the gene for full factor VIII in myoblasts and myotubes C ₂ Cl ₂
Obtaining myoblasts and myotubules of C ₂ Cl _{2 is} carried out as described above. Transfection is carried out using 6 μg of the plasmid encoding the full factor VIII human cDNA, as a complex with 5 or 15 μl of biotinylated adenovirus d1312, 0.5 or 1 μg of StpL and 7 or 6 μg of TfpL. Virus and DNA complexes are added to cells in DMEM containing 2% TES. After 4 hours of incubation at 37 ^° C., 3 ml of fresh DMEM medium containing 10% TES is added to each plate. After 18 hours, cells are harvested to determine the presence of factor VIII. Presented in FIG. 41 results are shown as milli-units in 24 hours on 1 • 10 ⁶ cells.

The use of adenovirus protein for DNA transfer. Wt300 adenovirus is grown in HeLa cells, purified and biotinylated as described above for adenovirus d1312. 1.2 ml of the virus is dialyzed using 3 x 300 ml of 5 mM MES medium containing 1 mM EDTA, pH 6.25, at a temperature of 4 ^° C. for 18 hours. Then centrifugation is carried out at 27 g for 30 minutes using a SW60 rotor. The supernatant was carefully removed and the residue resuspended in HBS buffer containing 40% glycerol. To the supernatant was added 20 mM HEPES buffer with pH 7.4 and 150 mM sodium chloride and both the sediment (containing the core of the virus and the bulk of the hexonic capsid, in Fig. 42 are designated as “heart”) and the fractions of the supernatant (containing the membranes, referred to in Fig. 42 as “envelopes.") are used to determine DNA transfer activity in murine Mov13 fibroblasts and HeLa cells.

The formation of a complex comprising DNA is as follows. Referring to FIG. 42 quantities of each fraction, i.e., the destroyed virus before centrifugation and the intact virus (expressed as μg protein according to the Bradford method), are diluted with 300 μl HBS buffer. Then add 3 μg of streptavidin-modified polylysine in 50 μl of HBS buffer, followed by incubation at room temperature for 30 minutes. 6 μg of pCMVL DNA was diluted with 100 μl of HBS buffer and added to the first solution, followed by incubation for 30 minutes. In conclusion, add 2 μg TfpL in 100 μl of HBS, followed by incubation for a further 30 minutes. To obtain samples containing only TfpL, 8 μg of TfpL in 170 μl of HBS was mixed with 6 μg of pCMVL in 330 μl of HBS at room temperature for 30 minutes. The indicated amounts of virus protein are diluted with 300 μl of HBS, after which they are added to the TfpL and DNA complexes. All samples are added to 5 • 10 ⁵ HeLa cells or Mov13 fibroblasts located in 6 cm diameter dishes containing 2 ml of DMEM medium containing 10% TES, after which the samples are left to stand for one hour. Then add 5 ml of fresh medium containing 10% TES, and after 20 hours, cells are collected to determine luciferase activity in light units. The results of the experiment are presented in FIG. 42, where graph A relates to an experiment using HeLa cells, and graph B to an experiment using Mov13 fibroplasts.

 As seen in FIG. 42, for both types of cells, a dose-dependent increase in DNA transfer activity is observed in the case of a shell-containing fraction (see Samples 4-6 in both graphs). If such an amount of biotinylated virus protein is included in complexes of TfpL and DNA that do not contain streptavidin-modified polylysine, then the degree of DNA transfer is close to the background level (see sample 3 in both graphs).

Example 28. Improved gene transfer when using triple complexes of DNA, including the conjugate of the galactose ligand and polylysine
a) Triple complexes comprising a peptide conjugate of influenza
It was found that in the presence of a polylysine-conjugated peptide comprising sequences derived from the N-terminus of the HA-2 subunit of influenza virus hemagglutinin in DNA and conjugate complexes of transferrin and polylysine leads to a significant improvement in gene transfer provided by transferrin / polylysine (see examples 13, 14). Similar DNA combination complexes, including a conjugate of polylysine and a galactose ligand, as well as a polylysine-modified influenza peptide (hereinafter “InflupL”; prepared as described in Examples 6, 13), are obtained by adding a ligand and polylysine conjugate to the pCMVL plasmid DNA to neutralize half the charge of DNA. The remaining charge is used to load the complexes with the conjugate of polylysine and the flu peptide. The addition of these DNA complexes, including the synthetic ligand (gal) 4, to the BNL CL.2 hepatocytes (transfection is carried out as described in Example 6g), resulting in the expression of the luciferase gene, which significantly exceeds the expression obtained with transferrin as a ligand (see Fig. 43). Expression is more than 500 times the expression found in control experiments using DNA complexes that contain the same amount of polylysine but do not contain influenza peptide (see also Fig. 43). The activity achieved by DNA combination complexes is also approximately 30 times greater than the activity achieved by DNA and (gal) 4pL complexes in the presence of chloroquine.

b) Triple complexes, including adenovirus conjugate
The complexes are prepared as follows. 2 μl, 6 μl and 18 μl, respectively, of biotinylated adenovirus d1312 (prepared as in Example 19) in an amount of 10 ¹² particles per ml in 50 μl of HBS are mixed with 100 ng, 160 ng and 480 ng, respectively, of 100 streptavidin-modified polylysine μl of HBS buffer, followed by incubation for 30 minutes. Then add a solution of 6 μg pCMVL in 200 μl of HBS and, after a further 30 minutes, a solution of 3.8 μg (gal) 4pL (prepared as in Example 6) or 7 μg of TfpL in 150 μl of HBS.

 Solutions of each DNA complex are added to 300,000 cells (ATCC TIB73, ATCC TIB74, ATCC TIB75, ATCC TIB76), which are grown in 6 cm diameter dishes containing DMEM medium with a high glucose content and 2% TES. Then they work as described above. Gene expression data (after 24 hours) is shown in FIG. 44.

Example 29. DNA transfer using conjugate transferrin and polylysine in the presence of free and conjugated rhinovirus
a) Preparations of rhinovirus HRV-2
400 μl of a rinovirus solution (approximately 30 μg) in HBS buffer (150 mM sodium chloride and 5 mM HEPES, pH 7.9) containing 10% glycerol was treated with 10 nmol NHS-LC biotin. After incubation at room temperature for 3 hours, the virus is separated from unincorporated biotin by extensive dialysis using an HBS buffer containing 40% glycerol at 4 ^° C. Obtained by growing in the presence of acridine orange (indicated in FIG. 45 as “acrid. ") the photosensitive rhinovirus is inactivated in a known manner.

b) Obtaining DNA complexes and transfection
1) Complexes of DNA and the conjugate of transferrin and polylysine are obtained by mixing a solution of 6 μg of plasmid DNA pCMVL in 330 μl of HBS buffer (150 mM sodium chloride, 20 mm HEPES, pH 7.3) with a solution of 8 μg TfpL290 in 170 μl of HBS. DNA complexes are mixed with 1.5 ml of DMEM containing 2% TES, and 0.14 μg - 3.5 μg of HRV-2 rhinovirus or HRV-2 inactivated rhinovirus. Mixtures are added to NIH 3T3 cells (300,000 cells per dish with a diameter of 6 cm). After 4 hours, the medium is replaced with 4 ml of fresh DMEM medium containing 10% TES. After 24 hours, cells were harvested to determine luciferase activity as described above. The results of the experiment are presented in FIG. 45A.

 2) DNA combination complexes, including the conjugate of transferrin and polylysine and the conjugate of rhinovirus and polylysine, are prepared as follows. A solution of 3.5 μg of biotinylated rhinovirus HRV-2 in 100 μl of HBS is mixed with 1 μg of streptavidin-modified polylysine in 100 μl of HBS. After 30 minutes at room temperature, the solution was mixed with 6 μg of plasmid DNA in 150 μl of HBS, incubated at room temperature for a further 30 minutes and then mixed with 6 μg of TfpL290 in 150 μl of HBS. Other complexes indicated in FIG. 45V. DNA complexes were mixed with 1.5 ml of DMEM containing 2% TES, and the mixture was added to NIH 3T3 cells (300,000 cells per dish with a diameter of 6 cm). Then proceed similarly to paragraph 1). The results of the experiment are presented in FIG. 45V.

 Example 30. Transfection of HeLa cells with combination complexes including ion-coupled adenovirus.

First, 30 μl of adenovirus d1312 (approximately 10 ⁹ plaque forming units) was mixed with 1 μg of polylysine with a molecular weight of 450, having an average length of 450 monomer units, in 170 μl of HBS. After 30 minutes at room temperature, 6 μg of pCMVL DNA in 170 μl of HBS was added to the mixture. After incubation for a further 30 minutes, the complexes are mixed with 9 μg of TfpL190 in 170 μl of HBS. An aliquot of the resulting complex (10% = 50 μl of a solution of 600 ng DNA or 1% = 5 μl of a solution of 60 ng DNA) is diluted with 1.5 ml of DMEM medium containing 2% TES, and then added to 300,000 HeLa cells. After 4 hours, add 2 ml of DMEM medium containing 20% TES. 24 hours after transfection, cells are harvested to determine luciferase activity as described above. The luciferase activity of the total extract is 29,115,000 light units (in the case of 600 ng DNA) or 1,090,000 light units (in the case of 60 ng DNA).

 Control experience.

6 μg of pCMVL DNA in 170 μl of HBS was mixed with 1 μg of polylysine with a molecular weight of 450 (having an average length of 450 monomer units) in 170 μl of HBS, and after 30 minutes, 9 μg of TfpL190 conjugate in 170 μl of HBS was added at room temperature. After a 30-minute incubation, the complexes are mixed with 30 μl of adenovirus d1312 (approximately 10 ⁹ plaque-forming units). An aliquot of the resulting complex (10% = 50 μl of a solution of 600 ng of DNA, or 1% = 5 μl of a solution of 60 ng of DNA) was diluted with 1.5 ml of DMEM medium containing 2% TES and added to 300,000 HeLa cells. After 4 hours, add 2 ml of DMEM medium containing 20% TES. Cell collection is carried out 24 hours after transfection. The determination of luciferase activity is carried out as described above. The total luciferase activity is 405,000 light units (in the case of 600 ng DNA) and 200 light units, respectively, in the case of 60 ng DNA.

 Example 31. Topical administration of DNA, adenovirus, and conjugate of transferrin and polylysine complex to rat liver.

a) Direct injection
The complexes are prepared as described in Example 19. They contain 200 μl of biotinylated adenovirus d1312, 6.4 μg streptavidin-modified polylysine, 48 μg pCMVL DNA and 48 μg TfpL290 conjugate. The complex is contained in 200,000 μl of HBS. A male rat of the Sprague-Dawley breed, weighing 240 g, is anesthetized with an avertine, after which 4 cm are heated. A solution of the complex is injected into the left lobe of the liver. Then the wound is closed. 48 hours after injection of the complex, the rat is euthanized and luciferase expression is determined. 5615 light units / mg protein of the liver homogenization product is determined in the injection zone. The total luciferase activity at the injection site is 370,600 light units.

b) The introduction of complexes into the liver through the bile drainage system
The complexes are prepared as follows. 200 μl of biotinylated adenovirus d1312 in 200 μl of HBS and 6.4 μg of streptavidin-modified polylysine in 400 μl of HBS are incubated at room temperature for 30 minutes, after which 48 μg of pCMVL DNA in 800 μl of HBS is added. After a 30 minute incubation, an additional 48 μg of TfpL conjugate in 900 μl of HBS is added. Male rats of the Spraque-Dawley breed weighing 250 g are anesthetized with an avertine, after which the abdominal cavity is opened by a midline incision. The intestine is moved to the left side of the body and a 27 G needle is inserted into the bile duct, connected to a 1 ml tube and syringe. The complexes are injected for 4 minutes, after which the needle is removed from the bile duct and the injection site, as well as the wound, is closed. After 30 hours, rats were sacrificed and samples were taken from various lobes of the liver to determine luciferase gene expression. The peak luciferase activity is 19,000 light units / mg protein, and the calculated total expression in the liver is 2.7 • 10 ⁶ light units.

 Example 32. Topical administration of DNA, adenovirus, and conjugate of transferrin and polylysine complex to the compressed mouse tail vein. The complexes are prepared as described in Example 19. These include 45 μl of biotinylated adenovirus d1312, 0.8 μg streptavidin-modified polylysine, 6 μg pCMVL DNA and 24 μg TfpL290 conjugate. A solution of the complexes in 150 μl of HBS is injected into the tail vein of a male mouse of C3H / He breed (2 months old) after preliminary anesthesia with avertin. Immediately after injection, the tail vein is squeezed for 20 minutes at the proximal and distal ends so that the solution of the complex was in that part of the tail vein into which it was injected and could not be washed with blood. 48 hours after injection, the mouse is sacrificed and the tail vein is prepared to determine luciferase expression. It is 2600 light units / 3 cm of the tail vein.

Example 33. Transfection of primary human melanoma cells. Primary melanoma cells are isolated from melanoma that was surgically removed from the patient due to mechanical destruction of the tumor in RPM1 1640 medium containing 5% TES, 2 mM glutamine and antibiotics, and subsequent tissue compression through steel sieve. Tumor cells are washed several times by centrifugation and subsequent re-suspension, and then cultured in T25 flasks. 24 hours after isolation, tumor cells are transfected with combination complexes, including 3 μl, 9 μl or 27 μl biotinylated adenovirus d1312 (1 • 10 ¹² virus particles / ml), 0.5 μg streptavidin-modified polylysine, 6 μg pCMVL DNA and 7 μg conjugate TfpL290, applied as a solution in 500 μl of HBS. 36 hours after transfection, cells are harvested and luciferase expression determined. The results of the experiment are presented in FIG. 46.

Example 34. Transfection of primary human fibroblasts. The skin tissue is fed into a 6 cm diameter petri dish containing DMEM medium, 2 mM glutamine, 20% TES and antibiotics. The tissue is carefully cut into small pieces with a surgical knife and cultured in the presence of 3 ml of medium for 5 days. The cells are then washed with fresh DMEM containing 2 mM glutamine, 10% TES and antibiotics, and cultured for a further 7 days, after which the cells are trypsinized and transferred to further culture in new petri dishes. When the cells are almost fused, they are again trypsinized and stored frozen. Before transfection, the cells are thawed and plated in 6 cm diameter petri dishes, in which they are cultured in DMEM containing 2 mM glutamine, 10% TES and antibiotics. The complexes for transfection are prepared as follows. 3 μl, 10 μl, 20 μl and 30 μl, respectively, of biotinylated adenovirus d1312 and 0.1 μg, 0.3 μg, 0.5 μg and 0.8 μg, respectively, of streptavidin-modified polylysine in 150 μl of HBS incubated for 30 minutes at room temperature. Then add 6 μg of plasmid pCMY-β-gal in 170 μl of HBS and the mixture is incubated for a further 30 minutes. Then add the TfpL conjugate in 170 μg of HBS in the following amounts: 7.8 μg in the case of a complex comprising 3 μl of adenovirus d1312, 7 μg in the case of a complex comprising 10 μl of adenovirus d1312, and 6 μg in the case of complexes comprising 20 μl and 30 μl of adenovirus d1312. After a 30-minute incubation, the complexes are added to the cells in 2 ml of DMEM medium containing 2 mM glutamine, 2% TES and antibiotics, and the cells are incubated at 37 ^° C for 4 hours, after which the medium is removed and cultivation is continued at 37 ^{° C.} C using DMEM medium containing 2 mM glutamine, 10% TES and antibiotics. After 48 hours, β-galactosidase expression is determined in the above manner.

 In the case of transfection with a complex comprising 3 μl of adenovirus d1312, 14% of the cells produce β-galactosidase. In the case of a complex comprising 10 μl of adenovirus d1312, 32% of positive cells are obtained, in the case of a complex comprising 20 μl of adenovirus d1312, 39% of positive cells, and in the case of a complex of 30 μl of adenovirus d1312, 64% of positive cells.

 Example 35. Gene transfer using non-viral endosomolytic agents.

a) Synthesis of membrane-destroying peptides
1) Peptide synthesis
The synthesis is carried out using an automatic apparatus of the ABI431A type by the solid phase method using 0.97 mmol / g para-alkoxybenzyl resin as a solid base and amino acids protected with 9-fluorenomethoxycarbonyl. An amino acid with a carboxyl group at the end is bound to the resin via symmetric anhydride. Subsequent amino acids are coupled with 1-hydroxybenzotriazole-dicyclohexylcarbodiimide. The following side chain protecting groups are used: (Trt) Asn, (Trt) Cys [(third-butyl) Cys in the case of the EALA and GLF peptides], (third-butyl) Glu, (Trt) His, (third-butyl ) Ser.

EALA: Trp Glu Ala Ala Leu Ala Glu Ala Leu Ala
Glu Ala Leu Ala Glu His Leu Ala Glu Ala Leu Ala Glu Ala Leu Glu Ala Leu Ala Ala
Gly Gly Ser Cys
GLF Gly Leu Phe Gly Ala Leu Ala Glu Ala Leu
Ala Glu Ala Leu Ala Glu His Leu Ala Glu Ala Leu Ala Glu Ala Leu Glu Ala Leu Ala Ala Gly Gly Ser Cys
GLF-II Gly Leu Phe Gly Ala Leu Ala Glu Ala Leu
Ala Glu Ala Leu Ala Glu Ala Leu Ala Glu Ala Leu Ala Glu Ala Leu Glu Ala Leu Ala Ala Gly Gly Ser Cys
GLF-delta Gly Leu Phe Glu Leu Ala Glu Ala Leu Ala
Glu Ala Leu Ala Glu Ala Leu Ala Glu Ala Leu Ala Glu Ala Leu Glu Ala Leu Ala Ala
Gly Gly Ser Cys
EALA-lnf Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu
Asn Gly Trp Glu Gly Leu Ala Glu Ala Leu Ala Glu Ala Leu Glu Ala Leu Ala Ala Gly
Gly ser cys
EALA-P50 Gly Leu Phe Glu Ala Ile Glu Gly Phe Ile Glu
Asn Gly Trp Glu Gly Leu Ala Glu Ala Leu Ala Glu Ala Leu Glu Ala Leu Ala Ala
Gly Gly Ser Cys
P50 Gly Leu Phe Glu Ala Ile Glu Gly Phe Ile Glu
Asn Gly Trp Glu Gly Met Ile Asp Gly Gly Gly Cys
The peptides are separated from the rubber and the side chain protecting groups [with the exception of (third-butyl) Cys] are removed by treating with a loaded 100 mg base peptide taken in an amount of 3 ml with a mixture of phenol, ethanedithiol, thionisole, water and trifluoroacetic acid in volume the ratio of 0.75: 0.25: 0.5: 0.5: 10 at room temperature for 90 minutes. Crude peptides precipitated in diethyl ether and washed twice. The EALA and GLF peptides protected by the tert-butyl mercapto group are dissolved in a small volume of 1M triethylammonium bicarbonate with a pH of 8, diluted to a volume of 100 mM triethylammonium bicarbonate and then purified by reverse phase liquid chromatography under pressure on a Nucleosil 500-5C4 column using 0.1 % trifluoroacetic acid and acetonitrile. Both peptides elute at approximately 50% acetonitrile. The free form of Cys-SH peptides is obtained by deprotecting Trt groups similarly to the above method. 5 mg of crude peptides are obtained, which are dissolved in 100 μl of a solution of 100 mM triethylammonium bicarbonate with pH 8 containing 1 μl of β-mercaptoethanol and purified by gel filtration on a Sephadex G-25 column (100 mM triethylammonium bicarbonate, 0.5 mM EDTA) ) and freeze-drying or ion-exchange chromatography on a Mono Q column (20 mM HEPES, pH 7.3, gradient: 0-3 M sodium chloride; the peptide was eluted at 1.5 M NaCl).

2) Modification of N- (hydroxyethyl) maleimide
After obtaining free mercapto form as a result of gel filtration on a Sephadex G-25 column (20 mM HEPES, pH 7.3, 0.5 mM EDTA) C-terminal mercapto group of GLF-delta, GLF-II, EALA-Inf, EALA- peptides P50 and P50 are blocked by reaction with a 1.3- to 10-fold molar excess of N- (hydroxyethyl) maleimide at room temperature for one hour. Excess maleimide is removed by gel filtration on a Sephadex G-25 column (100 mM triethylammonium bicarbonate, pH 8) and, after freeze drying, the peptides GLF-delta-mal, GLF-II-mal, EALA-Inf-mal, EALA-P50- mal, P50-mal as triethylammonium salt.

3) Modification of 2,2'-dithiobispiride otherwise
Peptides with a free mercapto group are reacted with 10 equivalents of 2,2'-dithiobispyridine in a 20 mM HEPES buffer pH 7.9 containing 0.5 mM EDTA, at room temperature overnight. Excess reagent is removed by gel filtration on a Sephadex G-25 column (100 mM triethylammonium bicarbonate, pH 8) or ion exchange chromatography on a Mono Q column (20 mM HEPES, pH 7.3, gradient: 0-3 M sodium chloride; elute the peptide at 1.5 M NaCl). The (2-pyridylthio) -Cys peptides GLF-delta-SSPy, GLF-H-SSPy, EALA-Inf-SSPy, EALA-P50-SSPy and P50-SSPy are obtained.

4) Dimerization of peptides
Homodimeric P50 (P50 dim) is obtained by reacting equimolar amounts of P50-Cys- (2-pyridylthio) and P50-Cys-SH in 20 mM HEPES, pH 7.3 at room temperature for 3 days. The reaction mixture was separated on a Mono Q HR-5/5 column (20 mM HEPES, pH 7.3, gradient: 0.09 - 3 M sodium chloride; dimeric P50 was eluted at 1.1 M NaCl). The heterodimeric GLF-SS-P50 is prepared in a similar manner by reacting the P50 peptide in the free mercapto form with the pyridylthio-modified GLF peptide.

b) Determination of liposome lysis
The ability of synthetic peptides to destroy liposomes is investigated by determining the release of a fluorescent dye from liposomes loaded with a self-extinguishing concentration of calcein. Liposomes are obtained from egg phosphatidylcholine by reverse phase evaporation from an aqueous phase containing 100 mM calcein, 375 mM sodium ions, 50 mM sodium chloride, having a pH of 7.3, and then passed through a polycarbonate filter with a pore size of 100 nm. Then the liposomes are purified by gel filtration on a Sephadex G-25 column using iso-osmotic buffer (200 mM sodium chloride, 25 mM HEPES, pH 7.3). To determine the lysis at different pH values, the basic liposome solution (6 μl / ml) was diluted with a buffer containing 400 mm sodium chloride and 40 mm sodium citrate. An aliquot of 100 μl was added to 80 μl of a different dilution aqueous solution of the peptide in a 96-well microtiter plate (final lipid concentration: 25 μmol), and after 30 minutes of incubation at room temperature, the fluorescence of calcein was determined at 600 nm (excitation at 490 nm). 100% lysis is obtained by adding 1 μl of a 10% solution of Triton X-100. Lysis units are calculated as the reciprocal of the concentration of the peptide at which 50% lysis is established (i.e., the volume (μl) of the liposome solution, which is subjected to 50% lysis per μg of peptide). Values below 20 units are the result of extrapolation. The results of the experiment are presented in FIG. 47, which shows that the GLF and EALA peptides exhibit the highest pH-specific activity.

c) Determination of hemolysis
Fresh human red blood cells are washed several times with HBS and resuspended in a buffer containing 300 mM sodium chloride and 30 mM sodium citrate at a concentration of 6.6 x 10 ⁷ / ml. An aliquot of 75 μl is added to 75 μl of a diluted aqueous solution of the peptide in a 96-well microtiter plate and incubated at 37 ^° C for one hour with constant agitation. After removing insoluble red blood cells by centrifugation at 1000 rpm for 5 minutes, 100 μl of supernatant is obtained, which is fed into a new microtiter plate, after which hemoglobin absorption is determined at 450 nm (background correction at 750 nm). 100% lysis is determined by adding 1 μl of a 10% solution of Triton X-100 before centrifugation. Hemolysis units are calculated as reciprocal values of the concentration of the peptide at which 50% lysis is detected (i.e., the volume (μl) of the erythrocyte solution that is subjected to 50% lysis per μg of peptide). Values below 3 units of hemolysis are the result of extrapolation. The results of the experiment are presented in FIG. 48, where it can be seen that the peptides P50 dim and EALA-P50 mal exhibit the highest pH-specific activity in cell lysis and / or release of large molecules, such as, for example, hemoglobin. Monomeric P50 mal and P50 SS-Py exhibit low activity. Melittin exhibits the highest activity, which, however, is not specific for acidic pH values.

g) Obtaining DNA-including combination complexes
6 μg of pCMVL DNA in 150 μl of HBS was mixed with 4 μg of TfpL290 conjugate in 150 μl of HBS, and after 30 minutes, 4 to 20 μg of poly (L) lysine with a molecular weight of 290 in 100 μl of HBS was added. After a further 30-minute incubation at room temperature, 0.3-30 μg of the peptide in 100 μl of HBS is added, and then incubated for another 30 minutes. The optimal amount of endosomolytic agent is preliminarily determined taking into account the results of gene transfer efficiency analysis (see table showing gene transfer to BNL CL.2 cells). The simultaneous addition of polylysine (pL) and an endosomolytic agent, as well as the use of a larger volume of the combination complex (final volume: 1.5 ml) lead to a comparable or better transfection efficiency. In this experiment, it was also found that non-peptide amphipathic substances deoxycholic acid and oleic acid can also improve DNA transfer.

g) Transfection of cells
Adherent cell lines (BNL CL.2 hepatocytes and NIH 3T3 cells, respectively) were grown in 6 cm diameter Petri dishes 1-2 days before transfection (medium: DMEM with 10% TES; 300,000 cells per dish). The medium is removed and 1.5 ml of DMEM medium containing 2% TES and 500 μl of the complex are added. Alternatively, 0.5 ml of DMEM containing 6% TES and 1.5 ml of the complex are used. After 4 hours of incubation, 2 ml of DMEM medium containing 18% TES is added. Alternatively, the transfection medium can also be replaced with 4 ml of DMEM medium containing 10% TES. 24 hours after transfection, cells are collected to determine luciferase activity in light units. The results of hepatocyte transfection of BNL CL.2 are presented in FIG. 49, showing total luciferase activity in transfected cells. Moreover, used in the experiment according to FIG. 49A. complexes are prepared as follows. 6 μg of pCMVL DNA in 250 μl of HBS was mixed with 4 μg of TfpL290 conjugate in 250 μl of HBS and after 30 minutes at room temperature 20 μg of poly (L) lysine with a molecular weight of 290 in 750 μl of HBS was added. After further incubation for 30 minutes at room temperature, the indicated amounts of peptides in 250 μl of HBS are added. After a further 30-minute incubation, the resulting complexes were mixed with 0.5 ml of DMEM containing 6% TES and added to 450,000 cells.

 The DNA complexes used in the experiment of FIG. 49B is prepared as follows. 6 μg of pCMVL DNA in 500 μl of HBS was mixed with 4 μg of TfpL290 conjugate in 250 μl of HBS and the solution was allowed to stand at room temperature for 30 minutes. A solution of 20 μg poly (L) lysine with a molecular weight of 290 in 500 μl of HBS was mixed with the indicated amounts of peptides in 250 μl of HBS and immediately added to the mixture of TfpL conjugate and DNA. After a 30-minute incubation at room temperature, the complexes are mixed with 0.5 ml of DMEM containing 6% TES and added to 450,000 cells. 24 hours after transfection, cells are harvested and luciferase activity is determined as described above.

 The results of experiments using NIH3T3 cells are presented in FIG. 50. Used in the experiments of FIG. 50A. and B. complexes are obtained in the same manner as complexes for the experiments of FIG. 49A. and B. FIG. 49 and 50 it can be seen that complexes including P50 dim are most active.

Example 36. Gene transfer using a synthetic non-viral peptide with oligilizine extension at the C-terminus
The peptide by the sequence Met Ala Gln Asp Ile Ile Ser Thr Ile Gly Asp Leu Val Lys Tip Ile Ile Asp Thr Val Asn Lys Phe Thr Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys are synthesized and purified by known techniques. This peptide is derived from the Staphylococcus aureus δ-toxin by the sequence Met Ala Gln Asp Ile Ile Ser Thr Ile Gly Asp Leu Val Lys Trp Ile Ile Asp Thr Val Asn Lys Phe Thr Lys Lys. This peptide has the ability to destroy membranes at acidic pH values if it is extended by an additional 10 lysine residues.

 6 μg of pCMVL DNA in 170 μl of HBS was mixed with 4 μg of TfpL290 conjugate in 170 μl of HBS. After 30 minutes at room temperature, approximately 3 μg of peptide in 170 μl of HBS was added. After a 30-minute incubation, the resulting complex is mixed with 1.5 ml of DMEM containing 2% TES and added to 450,000 BNL CL.2 hepatocytes. After 2 hours, add 2 ml of DMEM medium containing 20% TES. 24 hours after transfection, cells are harvested to determine luciferase activity as described above. In the total extract, it is 481,000 light units.

 Example 37. Transfection of hepatocytes in the presence of melittin peptides having an oligolysin tail at the C-terminus.

 Sequence peptides (N-terminus to C-terminus): Gly Ile Gly Ala Val Leu Lys Val Leu Thr Thr Gly Leu Pro Ala Leu Ile Ser Trp Ile Lys Arg Lys Arg Gln Gln Lys Lys Lys Lys Lys Lys Lys Lys Lys ( this peptide is hereinafter referred to as mel 1) and Gly Ile Gly Ala Val Leu Glu Val Leu Glu Thr Gly Leu Pro Ala Leu Ile Ser Trp lle Lys Arg Lys AArg Gln Gln Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys (an acid mutant that hereinafter referred to as mel 2) synthesized and purified by known techniques.

 6 μg of pCMVL DNA in 170 μl of HBS was mixed with 4 μg of TfpL290 conjugate in 170 μl of HBS and allowed to stand at room temperature for 30 minutes. Then add about 3 μg of the mel 1 peptide or 5 μg of the mel 2 peptide in 170 μl of HBS, and after a further 30-minute incubation, the resulting complex is mixed with 1.5 ml of DMEM containing 2% TES and added to 450,000 BNL CL cells. 2, which are pre-grown as described in example 36. After 4 hours, add 2 ml of DMEM medium containing 20% TES. 24 hours after transfection, cells are harvested and luciferase activity is determined as described above. In the total extract, luciferase activity is 9,200 light units (in the case of mel 1 peptide) and 9,400 light units (in the case of mel 2 peptide), respectively.

 Example 38. Expression of alpha interferon in HeLa cells.

5 • 10 ⁵ HeLa cells per 6 cm diameter Petri dish are transfected with the pAD-CMV1-IFN plasmid encoding human interferon alfa-2c under the control of the CMV enhancer / promoter (see DE 40 21 917 A; pADCMVI-IFN is obtained by cloning a Hindlll-insert XbaI / IFN-α 2c in pAD-CMV1). Samples of 6 μg of DNA in 330 μl of HBS were mixed with 8 μg of TfpL conjugate in 330 μl of HBS and the mixture was allowed to stand at room temperature for 30 minutes. Samples 6-10 contain only 4 μg of TfpL conjugate. After the first 30-minute incubation, aliquots of 20 μg P16pL in 160 μl of HBS are added to samples 6 and 7, and aliquots of 20 μg of polylysine with a molecular weight of 290 are added to samples 8, 9 and 10. After an additional 30-minute incubation, aliquots of 160 μl of buffer are added. HBS containing 10 μl (in the case of sample 8) or 50 μl (in the case of samples 9, 10) of free P16 (the synthesis of P16 and P16p1 is described in example 13). After an additional 30-minute incubation, samples are added to HeLa cells in 2 ml of DMEM containing 2% TES in the presence of the following additional substances. Samples 2, 7 and 10 contain 100 μmol of chloroquine ("Chl." In Fig. 51), samples 3, 4 - respectively 5 and 15 μl of adenovirus d1312 (1 • 10 ¹² particles / ml; "AdV" in Fig. 51) sample 5-15 μl of psoralen inactivated adenovirus d1312 ("inac. PS AdV" in Fig. 51). Samples 11, 12 and 13, containing only aliquots of the virus from samples 3, 4 and 5, serve as a control. Two hours after transfection, 5 ml of fresh DMEM medium containing 10% TES is added to the cells. 48 hours after transfection, the medium is replaced with 2 ml of fresh DMEM medium containing 10% TES. 72 hours after transfection, cells are collected to determine the interferon content using enzyme-linked immunosorbent assay. The experimental results, i.e., the interferon content in ng / ml, are presented in FIG. 51, where it is seen that the TfpL conjugate exhibits very low activity for the transfer of the interferon gene to these cells, which coincides with the results achieved with the use of luciferase and β-galactosidase genes. The presence of chloroquine results in a detectable signal (approximately 7 ng / ml; see sample 2). Adenovirus d1312 stimulates DNA transfer depending on the dose used (see Samples 3 and 4). Treatment of cells with comparable amounts of virus in the absence of a DNA-containing complex does not produce a detectable interferon signal (see Samples 11, 12). As a result of transfection with the endosomolytic peptide P16 according to example 13 in the form of a conjugate (see samples 6, 7) or as a substance connected by ionic route to the surface of the complex of DNA and TfpL conjugate (see samples 8, 9 and 10), a detectable level is reached production of interferon, which can be increased with the use of the conjugate of this peptide in the presence of chloroquine (see sample 7).

 Example 39. Gene transfer to B-lymphoblastoma cells.

 Conjugates of human immunoglobulin G or anti-human immunoglobulin G and polylysine are prepared by the method described below, the conjugation being carried out by known methods by introducing disulfide bridges after preliminary modification with succinimidylpyridyldithio propionate.

a) Obtaining conjugates of anti-human immunoglobulin G and polylysine containing 300 lysine residues (hereinafter: polylysine 300)
To a solution of 2 mg of anti-human goat immunoglobulin G in HBS buffer, consisting of 150 M sodium chloride and 20 mM HEPES buffer and having a pH value of 7.8, 14 μl of a 5 mM ethanolic solution of succinimidyl pyridylthio propionate was added. The mixture was left to stand at room temperature for 10 hours, after which it was subjected to gel filtration on Sephadex G-25 ion exchanger using 100 mM HEPES buffer having a pH value of 7.3 as eluent. Obtain 1.3 mg of anti-human immunoglobulin G, modified with 30 nmol of pyridyldithiopropionate residues. The polylysine 300 is modified by a similar method with succinimidyl pyridylthio propionate, and treatment with dithiothreitol and subsequent gel filtration is transferred to a form modified with free mercapto groups. A solution of 12 nmol of polylysine 300, modified with 29 nmol of mercapto groups, in 0.3 ml of HBS buffer with the exception of oxygen was mixed with the above modified anti-human immunoglobulin G, and the resulting mixture was left to stand at room temperature overnight. Then, by adding 5 M sodium chloride, the content of sodium chloride in the reaction mixture was adjusted to 0.6 M. The conjugates were isolated by ion exchange chromatography on Mono S HR 5/5 ionite, Pharmacia, Sweden. Dialysis using 25 mM HEPES buffer, pH 7.3, gives the corresponding conjugates consisting of 0.33 mg of anti-human immunoglobulin G, modified 4 nmol of polylysine 300, and the molar ratio of anti-human immunoglobulin G and polylysine 300 is 1 : 2.

b) Obtaining conjugates of human immunoglobulin G and polylysine 300
To a solution of 19.5 mg (122 nmol) of I-4506 antibodies from Sigma, DE, in 2 ml of HBS buffer, 39 μl of a 1 mM ethanolic solution of succinimidyl pyridylthio propionate was added. The mixture was allowed to stand at room temperature for 2.5 hours, after which it was subjected to gel filtration on Sephadex G-25 ionite using 100 mM HEPES buffer, pH 7.9, as an eluent, to give 19 mg (119 nmol) human immunoglobulin G modified with 252 nmol of pyridyldithiopropionate residues. Modification of polylysine 300 with succinimidyl pyridylthiopropionate was carried out by a similar method, and treatment with dithiothreitol followed by gel filtration was transferred to a form modified with free mercapto groups. A solution of 119 nmol of polylysine 300, modified 282 nmol of mercapto groups, in 1 ml of HBS buffer with the exception of oxygen was mixed with the above modified anti-human immunoglobulin G, and the resulting mixture was allowed to stand at room temperature overnight. Then, by adding 5 M sodium chloride, the sodium chloride content in the reaction mixture was adjusted to approximately 0.6 M. The conjugates were isolated by ion exchange chromatography on Mono S brand ionite from Pharmacia, Sweden, using 50 mM HEPES buffer, pH 7.3 as eluent. with a salt gradient of 0.6 M - 3 M sodium chloride. Dialysis using a HEPES buffer, pH 7.3, gives the corresponding conjugates consisting of 9 mg (57 nmol) of an antibody modified with 90 nmol of polylysine 300. The molar ratio of antibody to polylysine 300 is 1: 1.6.

c) Complex formation and transfection
The complexes are prepared as follows: 30 μl of biotinylated adenovirus d1312 (10 ¹² particles per ml) in 50 μl of HEPES buffer is mixed with 800 ng of streptavidin-modified polylysine in 100 μl of HBS buffer. After incubation for 30 minutes, a solution of 9 μg pCMVL DNA in 200 μl HBS buffer is added. Incubate again for 30 minutes, after which a solution of 5.1 μg of polylysine 450 (pL), 10.2 μg of TfpL conjugate, 12 μg of human immunoglobulin G and polylysine conjugate or 10 μg of anti-human immunoglobulin G and polylysine conjugate in 150 are added. μl of HBS buffer. DNA complexes are added to 10 ⁶ B-lymphoblastoma cells (cells are obtained from peripheral mononuclear human blood cells as a result of immortalization with Epstein and Barr virus grown in a 24-well cup in 1 ml of RPMI 1640 medium with 2% TES). Further processing of cells and experiments to determine the activity of luciferase is carried out as described above. The obtained gene expression data (luciferase activity in light units) are shown in FIG. 52.

 Example 40. Transfection with combined complexes of adenovirus and polylysine conjugate and low density lipoprotein conjugate and polylysine.

 a) Obtaining conjugates of low density lipoprotein and polylysine.

 To a solution of 10 mg (14.3 nmol) of low density lipoprotein (conditional reduction in FIG. 53: LDL, product L-2139 with a molecular weight of 3,500,000 from Sigma, DE, particle diameter of about 26 nm) in 2 ml of HBS buffer, 143 μl was added 10 mm ethanol solution of succinimidyl pyridylthio propionate (1.43 μmol), and the resulting mixture was left to stand at room temperature for 2 hours. As a result of gel filtration on Sephadex G-25 ion exchange resin (14 x 140 mm column) using HBS buffer as an eluent, 3.2 ml of a solution of approximately 10 mg of low density lipoprotein modified with 0.70 μmol of pyridyldithiopropionate residues are obtained. 1.2 ml of 5 M sodium chloride was added to this solution in order to prevent the formation of a precipitate, which would otherwise have occurred with the subsequent addition of polylysine. Polylysine 300 is modified in the manner described above with succinimidyl pyridylthiopropionate and, by treatment with dithiothreitol and subsequent gel filtration, is converted to a form modified with mercapto groups. A solution of modified low-density lipoprotein in an argon atmosphere is mixed with a solution of 0.33 μmol of polylysine 300, modified 0.76 μmol of mercapto groups, in 4 ml of 2 M sodium chloride and 0.5 M HEPES buffer, pH 7.9, then the reaction mixture left to stand at room temperature for 48 hours. The resulting reaction solution was diluted by adding sterile water to a volume of 32 ml (after which the concentration of sodium chloride was approximately 0.5 M), and it was purified by ion exchange chromatography on a Biorad Macroprep S column, 10 x 100 mm, using 20 mM buffer as eluent HEPES with a gradient of 0.2-3 M sodium chloride. Product fractions are obtained at a salt concentration of 1.8-2.2 M and are collected. As a result of dialysis using HBS buffer, conjugates are obtained consisting of 2.35 mg (3.4 nmol) of low density lipoprotein modified with 190 nmol of polylysine, which corresponds to 7.5 mg of the free basic form of polylysine. Thus, low density lipoprotein modified 55 polylysine residues.

b) Preparation of complexes and transfection
18 μl of biotinylated adenovirus d1312 / 16 was diluted by adding HBS buffer to a volume of 100 μl. In addition, HBS buffer is added to 1.2 μg of streptavidin-modified polylysine to a volume of 100 μl and mixed with the indicated adenovirus. After 30 minutes, add 150 μl of HBS buffer containing 6 μg of pCMVL DNA. After a further 30 minutes, add 300 μl of HBS buffer containing 4 μg of LDLpL conjugate (with a polylysine content of 20 μg). The resulting solution was stirred with 1.5 ml of DMEM (10% TES) and added to 3 • 10 ⁵ primary melanoma cells obtained in Example 33 and designated HMM1 and HMM4, located in an incubation dish with a diameter of 6 mm. Further cell growth operations and determination of luciferase activity are carried out as described in Example 33. Gene expression data are shown in FIG. 53; part A) of FIG. shows the results obtained in experiments with melanoma cells HMM1. All experiments were performed with AdpL conjugates not shown in this FIG. In part B) of FIG. shows the results obtained in experiments using cells of melanoma HMM4. And in this case, all experiments are carried out with AdpL conjugates, which, however, are given only separately in the last column ("d1312 / 16" refers to a special preparation of the virus). In the experiment, the results of which are shown in the second column, low-density lipoprotein is used in a 25-fold excess, which, due to competition with the low-density lipoprotein receptor, leads to a decrease in gene transfer efficiency.

Example 41. In vivo gene transfer into rat epithelial cells of the respiratory tract using combined complexes
a) Obtaining combined complexes of DNA and conjugates TfpL and AdVpL
By combining 8 μg of the human transferrin conjugate and polylysine (hTfpL) in 150 μl of HBS buffer containing 150 mm of sodium chloride and 20 mm of HEPES buffer, pH 7.3, with 6 μg of pCMVL DNA in 350 μl of HBS buffer, complexes of DNA and said conjugate are obtained which are incubated at room temperature for 30 minutes. Conjugates of adenovirus and polylysine are obtained analogously to the method described in example 19c), using an adenovirus inactivated by treatment with psoralen and UV rays. Combined complexes of DNA and hTfpL and AdVpL conjugates are prepared according to the method described in Example 23c).

b) Introduction of complexes along the intratracheal pathway into a living organism
To carry out this experiment, Sigmodon hispidus rats (the so-called "Cotton Rat") are used, which are suitable animals for the study of human adenoviral lung diseases. Animals are anesthetized with methoxyflurane. A vertical incision is made on the ventral side of the throat, after which the trachea is dissected. Then the rats are laid on their sides at an angle of 45 ^° , and 250-300 μl of complexes containing 3 μg of DNA are injected directly into the trachea. After the expiration of the intervals indicated in FIG. 54, the animals are sacrificed by giving carbon dioxide, and the trachea and its associated lungs are removed after preliminary washing in situ with a cold phosphate-containing sodium chloride solution. To determine luciferase activity, lung tissue is homogenized in a buffer medium, extraction lysates are standardized for total protein content, and luciferase gene expression is measured as described above. The indicated light units refer to 1.250 μg of total protein obtained from lung lysates. The experiments are carried out 3-4 times, the results are average values ± average deviation.

Claims (75)

 1. Transfection composition for higher eukaryotic cells, comprising a complex of nucleic acids and substances with affinity for nucleic acids, characterized in that it additionally contains an endosomolytic agent in an effective amount.  2. Transfection composition for higher eukaryotic cells according to claim 1, characterized in that the substance with an affinity for nucleic acid is associated with a factor that promotes absorption by the cell.  3. Transfection composition for higher eukaryotic cells according to claim 1, characterized in that the endosomolytic agent is a virus or a component of the virus.  4. Transfection composition for higher eukaryotic cells according to claim 1, characterized in that the endosomolytic agent comprises a nucleic acid binding domain.  5. Transfection composition for higher eukaryotic cells according to claim 1, characterized in that the endosomolytic agent is a component of a complex of nucleic acids and substances with affinity for nucleic acids.  6. Transfection composition for higher eukaryotic cells according to claim 5, characterized in that the complex includes a factor promoting cell uptake.  7. Transfection composition for higher eukaryotic cells according to claim 6, characterized in that the factor promoting the absorption of the cell is associated with a substance with an affinity for nucleic acid.  8. Transfection composition for higher eukaryotic cells according to claim 5, characterized in that the endosomolytic agent is covalently linked to a substance with an affinity for nucleic acid.  9. Transfection composition for higher eukaryotic cells according to claim 5, characterized in that the endosomolytic agent is non-covalently associated with a substance with an affinity for nucleic acid.  10. Transfection composition for higher eukaryotic cells according to claim 9, characterized in that the endosomolytic agent is associated with a substance with an affinity for nucleic acid through a biotin-streptavidin bridge.  11. Transfection composition for higher eukaryotic cells according to claim 9, characterized in that the endosomolytic agent is ionically linked to a substance with an affinity for nucleic acid.  12. Transfection composition for higher eukaryotic cells according to claim 4, characterized in that the endosomolytic agent is associated with a nucleic acid directly through a nucleic acid binding domain.  13. Transfection composition for higher eukaryotic cells according to claim 3, characterized in that as an endosomolytic agent contains a virus that is infectious for an individual other than a person.  14. Transfection composition for higher eukaryotic cells according to claim 3, characterized in that it contains an adenovirus as an endosomolytic agent.  15. Transfection composition for higher eukaryotic cells according to 14, characterized in that the adenovirus is avian.  16. Transfection composition for higher eukaryotic cells according to clause 15, wherein the adenovirus is chicken adenovirus CELO.  17. Transfection composition for higher eukaryotic cells according to 14, characterized in that the adenovirus is a mutant.  18. Transfection composition for higher eukaryotic cells according to 17, characterized in that the adenovirus is a mutant incapable of replication.  19. Transfection composition for higher eukaryotic cells according to p, characterized in that the adenovirus has at least one mutation and / or deletion in the E1A zone.  20. Transfection composition for higher eukaryotic cells according to 14, characterized in that the adenovirus is inactivated.  21. Transfection composition for higher eukaryotic cells according to claim 20, characterized in that the adenovirus is inactivated by short-wave UV rays.  22. Transfection composition for higher eukaryotic cells according to claim 20, characterized in that the adenovirus is an inactivated treatment with UV rays and psoralen.  23. Transfection composition for higher eukaryotic cells according to claim 20, characterized in that the adenovirus is an inactivated formaldehyde.  24. Transfection composition for higher eukaryotic cells according to claim 3, characterized in that as an endosomolytic agent contains at least one adenovirus protein.  25. Transfection composition for higher eukaryotic cells according to claim 3, characterized in that it contains picornavirus as an endosomolytic agent.  26. Transfection composition for higher eukaryotic cells according A.25, characterized in that as picornavirus contains rhinovirus.  27. Transfection composition for higher eukaryotic cells according to p, characterized in that the rhinovirus is inactivated.  28. Transfection composition for higher eukaryotic cells according to claim 3, characterized in that as an endosomolytic agent contains an endosomolytic viral peptide, which can be modified.  29. The transfection composition for higher eukaryotic cells according to claim 28, wherein the endosomolytic viral peptide is an influenza hemagglutinin HA2 peptide.  30. The transfection composition for higher eukaryotic cells according to clause 29, wherein the peptide has the sequence Gly-Leu-Phe-Clu-Ala-Ile-Gly-Phe-Ile-Glu-Asn-Gly-Trp-Glu-Gly- Met-Ile-Asp-Gly-Gly-Gly-Cys.  31. The transfection composition for higher eukaryotic cells according to clause 29, wherein the peptide has the sequence Gly-Leu-Phe-Gly-Ala-Ile-Ala-Gly-Phe-Ile-Glu-Asn-Gly-Trp-Glu- Gly-Met-Ile-Asp-Gly-Gly-Gly-Cys.  32. The transfection composition for higher eukaryotic cells according to claim 1, characterized in that the endosomolytic peptide is a non-viral natural or synthetic peptide having the ability to modify.  33. The transfection composition for higher eukaryotic cells according to claim 28 or 32, wherein the peptide has a nucleic acid binding domain.  34. Transfection composition for higher eukaryotic cells according to claim 2 or 6, characterized in that the transferring factor is a transferin.  35. The transfection composition for higher eukaryotic cells according to claim 2 or 6, characterized in that the hepatocyte ligand is a contributing factor to cell uptake.  36. The transfection composition for higher eukaryotic cells according to claim 2 or 6, characterized in that the ligand for the azialoglycoprotein receptor is a contributing factor to cell uptake.  37. The transfection composition for higher eukaryotic cells according to claim 2 or 6, characterized in that the factor promoting the uptake of the cell is tetra-galactose polylysine.  38. A nucleic acid complex suitable as a component of a transfection composition according to any one of claims 4 to 37, characterized in that it comprises at least one nucleic acid to be expressed in a cell, an endosomolytic agent that initially has a nucleic acid binding domain or linked to a nucleic acid affinity.  39. The complex according to § 38, characterized in that it further comprises a factor that promotes cell uptake associated with a substance with an affinity for nucleic acid.  40. The complex of claim 38, wherein the nucleic acid is therapeutically active.  41. The complex of claim 40, wherein the nucleic acid comprises at least one DNA molecule active in gene therapy.  42. The complex according to paragraph 41, wherein the DNA molecule encodes a cytokine.  43. The complex of claim 40, wherein the nucleic acid has a nucleotide sequence that allows transcription of an RNA molecule that inhibits cell function.  44. The complex of claim 38, wherein the nucleic acid affinity is an organic polycation.  45. The complex according to claim 44, wherein the polycation is polylysine.  46. The complex according to claim 38 or 39, characterized in that the endosomolytic agent and the cell-promoting factor are associated with the same substance with an affinity for nucleic acid.  47. The complex according to claim 46, wherein the endosomolytic agent and the factor promoting cell uptake are associated with polylysine.  48. The complex according to item 47, wherein it further includes non-covalently bound polylysine.  49. The conjugate suitable as a component of the complex according to any one of paragraphs. 38-48, characterized in that it includes an endosomolytic agent associated with a substance with an affinity for nucleic acid.  50. The conjugate according to § 49, wherein the endosomolytic agent is covalently linked to a substance with an affinity for nucleic acid.  51. The conjugate of claim 49, wherein the endosomolytic agent is non-covalently linked to a nucleic acid affinity.  52. The conjugate according to claim 51, wherein the endosomolytic agent is bound to the substance through a biotin-streptavidin bridge.  53. The conjugate according to paragraph 51, wherein the endosomolytic agent is ionically bound to the substance.  54. The conjugate according to claim 49, wherein the endosomolytic agent is a virus or a component of the virus.  55. The conjugate according to item 54, wherein the virus is infectious for an individual other than a person.  56. The conjugate according to item 54, wherein the virus is an adenovirus.  57. The conjugate according to item 54, wherein the adenovirus is avian.  58. The conjugate according to clause 57, wherein the adenovirus is chicken CELO adenovirus.  59. The conjugate of claim 56, wherein the adenovirus is a mutant.  60. The conjugate according to § 59, wherein the adenovirus is a replication-inconsistent mutant.  61. The conjugate according to claim 60, wherein the adenovirus has at least one mutation and / or deletion in the E1A region.  62. The conjugate of claim 56, wherein the adenovirus is inactivated.  63. The conjugate according to claim 62, wherein the adenovirus is inactivated by short-wave UV rays.  64. The conjugate of claim 62, wherein the adenovirus is an inactivated treatment with ultraviolet light and psoralen.  65. The conjugate of claim 62, wherein the adenovirus is inactivated formaldehyde.  66. The conjugate according to item 54, wherein the component of the virus is an adenovirus protein.  67. The conjugate according to item 54, wherein the virus is a picornavirus.  68. The conjugate according to Claim 67, wherein the picornavirus contains rhinovirus.  69. The conjugate according to claim 49, wherein the endosomolytic agent is an endosomolytic viral peptide that can be modified.  70. The conjugate of claim 69, wherein the endosomolytic viral peptide is a influenza hemagglutinin HA2 peptide.  71. The conjugate according to claim 70, wherein the peptide has the sequence Gly-Leu-Clu-Ala-Ile-Gly-Phe-Ile-Glu-Asn-Gly-Trp-Glu-Gly-Met-Ile-Asp-Gly -Gly-Gly-Cys.  72. The conjugate according to claim 70, wherein the peptide has the sequence Gly-Leu-Phe-Gly-Ala-Ile-Ala-Gly-Phe-Ile-Glu-Asn-Gly-Trp-Glu-Gly-Met-Ile -Asp-Gly-Gly-Gly-Cys.  73. The conjugate according to claim 49, wherein the endosomolytic agent is a non-viral natural or synthetic peptide having the ability to modify.  74. An endosomolytic peptide suitable as a component of the transfection composition according to claim 33, characterized in that it comprises an endosomolytic domain and a nucleic acid binding domain.  75. The endosomolytic peptide of claim 74, wherein the nucleic acid binding domain is an oligolysin tail. Priority on points:
09/30/91 - for PP. 5; 8-10; 22, 39, 46-48, 50-52, 54, 56, 59-64, 69-71 (application 07/767788);
09/30/91 - according to claims 2, 3, 6, 7, 14, 17-21, 23, 28-30, 34, 36, 40, 41, 43-45, 65, 66, 67 (application 07/768039) ;
04/07/92 - according to claims 13, 15, 16, 37, 55, 57, 58, 72;
09/02/92 - according to claims 1, 4, 11, 12, 24, 25-27, 31-33, 35, 38, 42, 49, 53, 68, 73-75.

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