HRP920602A2 - Composition for introducing nucleic acid complexes into higher eucaryotic cells - Google Patents

Abstract

A higher eukaryotic cell transfection composition, comprising nucleic acid complexes and a substance having an affinity for a nucleic acid and optionally incorporating a factor, contains an endosomolytic agent, e.g. a virus or a viral component that can be conjugated. The endosomolytic agent, which may be part of the nucleic acid complex, is incorporated into the cells together with the complex and releases endosome contents into the cytoplasm, thereby enhancing gene transfer capacity. Also disclosed are pharmaceutical compositions, transfection kits and methods for introducing nucleic acid into multiple eukaryotic cells by treating the cells with composition.

Description

The invention is from the field of DNA technology. The invention particularly relates to new compositions that can be used to introduce nucleic acids into more eukaryotic cells.

There is a need for an effective system for introducing nucleic acid into living cells, especially in gene therapy. Genes are transferred into cells for the purpose of achieving "in vivo" synthesis of therapeutically effective genetic products, for example, replacement of a defective gene in the case of a genetic deficiency. "Conventional" gene therapy is based on the principle of achieving a permanent cure with a single treatment in which therapeutically effective DNA ( or mRNA) is administered as a drug ("genetic therapeutic agent") on one or, if necessary, several times in a row. Examples of genetically caused diseases, in which gene therapy represents a promising approach, are: hemophilia, beta-thalassemia and "severe combined immune deficiency", SCID (Severe Combined Immune Definiency), a syndrome caused by a genetically introduced deficiency of the enzyme adenosine deaminase. Other applications are possible in immune regulation, whereby humoral or intracellular immunity is achieved by administering a functional nucleic acid, which acts as a masked protein antigen or as an unmasked protein antigen, which can be considered as vaccination. Other examples of genetic defects, in which two nucleic acids can be acid, as a replacement for a damaged gene, for example in a form individually determined for a special request, are represented by muscular dystrophy (dystrophin gene), cystic fibrosis (cystic fibrosis gene that regulates transmembrane conductance), hypercholesterolemia (LDL receptor gene). Gene therapy methods are also potentially useful when it is necessary to synthesize hormones, growth factors or proteins with a cytotoxic or immuno-modulating effect in the body.

Gene therapy appears as a hope in the treatment of cancer, by giving the so-called "vaccine cancer". To increase the immunogenicity of tumor cells, they are altered to become more antigenic or to produce certain immune-modulating substances, eg cytokines, to stimulate an immune response. This is achieved by transfecting cells with DNA that indicates a cytokine, eg IL-2, IL-4, IFN gamma, TNF alpha. To date, most gene transfer into autologous tumor cells is achieved via retroviral vectors.

The technologies, which are the most advanced so far in the delivery of nucleic acids in gene therapy, use retroviral systems for gene transfer into cells (Wilson I et al., 1990, Kasid I et al., 1990). However, the use of retroviruses is problematic, because it brings, at least to a small extent, the risk of side effects, such as virus infection (by recombination with endogenous viruses or contamination with helper viruses and then possible mutation into a pathogenic form) or the formation of cancer. Furthermore, constant the transformation of body cells in the patient, which is achieved by retroviruses, is not desirable in any case, because it can complicate the treatment in the opposite sense, for example, if side effects occur. In addition, with this type of therapy, it is difficult to achieve a high enough titer to infect enough cells.

Nucleic acids, as therapeutically effective substances, can also be used to inhibit certain cellular functions, eg RNA and DNA acids have been shown to be effective for selective inhibition of specific gene sequences. Their mode of action allows them to be used as therapeutic agents to stop the expression of some genes "in vivo" (such as unspecified oncogenes or viral genes). It has already been shown that short oligonucleotides can be introduced into cells and that they can exert their inhibitory effect there (Zamecnik I et al., 1986), even with a low intracellular concentration, caused, among other things, by their limited uptake by the cell membrane as a result of a strong negative charge of nucleic acids. Another approach to selective gene inhibition is the use of ribozymes. Here, too, it is necessary to ensure the highest possible concentration of active ribozymes in the cell, with transport in the cell being one of the limiting factors.

The use of gene therapy to achieve intracellular immunity includes the translation of genes that protect against viruses, i.e. the so-called "protective genes", eg transdominant mutants of genes that mean viral proteins, or DNA molecules, that mean t. the so-called RNA decoys. Therefore, there is a need for methods of enabling the transfer and expression of DNA in a cell.

Different techniques of gene transformation in mammalian cells "in vitro" are known, but their use "in vivo" is limited (these techniques include introduction of DNA using liposomes, electroporation, microinjection, cell fusion, DEAE-dextran or calcium phosphate precipitation methods).

More recently, recombinant viral vectors have been developed that contribute to gene transfer by utilizing the efficient entry mechanisms of their parent viruses; this strategy was used in the construction of recombinant retroviral and adenoviral vectors with the aim of achieving highly efficient gene transfer "in vitro" and "in vivo" (Berkner, 1988). Despite their efficiency, these vectors are subject to limitations regarding the size and construction of the DNA being transferred. Furthermore, these agents pose a safety risk in view of the simultaneous transfer of viable viral elements of the original virus gene.

In order to circumvent these limitations, alternative strategies for gene transfer have been developed, which are based on the mechanisms used by cells to transfer macromolecules. One example of this is the transfer of genes into a cell by a particularly efficient pathway of receptor-mediated endocytosis (Wu and Wu, 1987, Wagner et al., 1990, EP-A1 0388 758, the disclosure of which is published herein). This approach uses bifunctional molecular conjugates that have a DNA binding domain and a domain specific for cell surface receptors (Wu and Wu, 1987, Wagner et al. 1990). If the recognition domain is recognized by a cell surface receptor, the conjugate is expressed via receptor-directed endocytosis, in which the DNA binding to the conjugate is also transferred. Using this method, it is possible to achieve gene transfer rates at least as good as those achieved by conventional methods (Zenke et al., 1990). Furthermore, it was shown that nucleic acid activity, eg ribozyme inhibitory effect, is not weakened by the transport system.

PCT application WO 91/17773 relates to a delivery system for nucleic acids with specific activity for T-cells. This system uses cell surface proteins of T-cell origin, eg CD4, the receptor used by the HIV virus. The nucleic acid to be introduced is complexed with a protein-polycation conjugate, whose protein component, i.e. recognition domain, a protein capable of binding to a T-cell surface protein, eg, CD4, and cells displaying this surface protein are brought into contact with the resulting protein-polycation/nucleic acid complexes. It has been shown that DNA transported into the cell by this system comes into the cell.

One feature common to both systems (inventions) is that they use specific cell functions that enable or facilitate the transfer of nucleic acid into the cell. In both cases, the acceptance mechanisms occur with the participation of recognition domains, which are referred to as "intrinsic factors" within the scope of the present invention. This term denotes ligands, which, as being specific for the cell type in a narrower or broader sense, bind to the cell surface and are drawn inside, possibly with the participation of other factors (eg cell surface proteins). (In the case of the two above-mentioned inventions, the internalizing factor is transferrin or a protein that binds to a T-cell surface antigen, eg an anti-CD4 antibody). The internalizing factor is conjugated with a polycationic substance which, based on its affinity with nucleic acids, forms a strong connection between the internalizing factor and nucleic xylene. (Substances of this type are referred to hereafter as "substances with affinity for nucleic acid" or, with respect to DNA, "DNA binding domain". If a substance of this type creates a bond between nucleic acid and an internalizing factor, it is referred to hereafter as a "factor connection").

During these two inventions, optimal nucleic acid uptake into the cell was achieved when the ratio of the conjugate to the nucleic acid was such that the internalizing factor-polycation/nucleic acid complexes were approximately electroneutral. Based on these observations, methods using internalizing factor-binding factor/nucleic acid complexes to introduce nucleic acid into more eukaryotic cells have been improved.

A method of improving the efficiency of systems in which the acceptance of nucleic acids is performed using internalizing factors was described by Wagner et al., 1991. In this method, the amount of nucleic acid accepted into the cell did not decrease, if part of the transferrin polycation conjugate was replaced by non-covalently bound ("free") polycation. In some cases, it can even increase DNA uptake significantly. Research on the molecular state of transferrin-polycation-plasmid DNA complexes produced with optimal DNA/conjugate ratios showed that plasmid DNA in the presence of conjugate condenses into toroidal structures (similar to paper clips) with a diameter of about 80 to 100 nm.

Experiments performed with cell-bound proteins as an internalizing factor gave similar results.

The addition of "free" substances with affinity for nucleic acid also results in an increase in the efficiency of introduction of the system, even when other binding factors are used.

The complexes described by Wagner et al., 1991a, which are taken up into higher eukaryotic cells via endocytosis by internalizing factor, contain nucleic acid complexed with a conjugate of internalizing factor and binding factor. In addition, the complexes contain one or more substances with affinity for the nucleic acid, which may be identical to the binding factor, in a non-covalent form, so that the internalization and/or expression of the nucleic acid achieved by the conjugate is increased, which appears to be caused primarily by a condensing effect , but it is possible that it is also caused by other mechanisms.

Even if the expression rates of the introduced nucleic acid could be increased by this method, there are still limitations. The practicality of this system in a given context is not only determined by the presence of a cell surface receptor relevant to the system; limitations related to the use of this system are probably the result of the fact that conjugate-DNA complexes internalized in endosomes enter lysosomes, where they are degraded by enzymes. In order to increase the proportion of nucleic acid that reaches the cell nucleus and is expressed there, which was desired, attempts were made in experiments that preceded this invention, to transfect cells in the presence of substances that inhibit enzymatic activity in lysosomes, the so-called lysosomotropic substances. This strategy achieved increased expression of the transferred DNA; however, the reactions achieved were highly variable, depending on the substance used; selected lysosomatotropic substances led to an increase in gene transfer, while others inhibited it. Thus, for example, efficient DNA transfer was found to depend on the presence of the weak base chloroquine (Zenke et al. 1990, Cotten et al. 1990). This effect achieved by chloroquine may not, at least exclusively, be caused by the fact that chloroquine increases the pH in lysosomes; it was found on the basis of numerous different experiments that other substances, which, like chloroquine, have the ability to change pH, such as monensin, ammonium chloride or methylamine, cannot replace chloroquine, and in some experiments some of these substances even showed an inhibitory effect. It was also found that different target cells show different responses to the same substances that have a lysosomotropic effect.

Since the transfer of genes by a physiological route, as represented by receptor-directed endocytosis with the use of nucleic acid complexes, has the main advantages (non-toxic mechanism of passage through the cell membrane; the possibility of providing biologically active nucleic acids, such as nucleic acids that specifically inhibit genes, or cellular function, on a repeated or continuous basis; the possibility of cell-specific targeting; the possibility of producing conjugates in large quantities), there is a need to make this system more effective.

Description of images

Sl. 1: Effect of adenovirus infection on gene transfer using transferrin-polylysine conjugates.

Sl. 2: Dosing effect of the conjugate-DNA-complex.

Sl. 3: Amplification of transferrin-polylysine-directed gene transfer by adenovirus occurs through receptor-directed endocytosis

A) Effect on complexed DNA

B) Effect on receptor-bound DNA

C) Effect on gene transfer via transferrin-polylysine conjugate.

Sl. 4: Effect of adenovirus infection on gene transfer using transferrin-polylysine conjugates and selected cell lines.

Sl. 5: Investigations whether the amplification of gene expression depends on gene transfer or transactivation.

A) Cell line K562

B) K562 10/6 cell line constitutively expressing luciferase.

Sl. 6: Tetra-galactose peptide-polylysine conjugate.

Sl. 7: Transfection of HepG2 cells in the presence of adenovirus.

Sl. 8: Transfection of HepG2 cells in the presence of adenovirus.

Sl. 9: Transfection of TIB73 cells

A) Comparative values with chloroquine

B) In the presence of adenovirus.

Sl. 10: Transfection of T cells in the presence of adenovirus:

A) H9 cells

B) Primary lymphocytes.

Sl. 11: UV-inactivation of adenoviruses:

A) Enhancement of gene transfer effect in HeLa cells with UV-inactivated viruses

B) Comparison of UV-inactivation with the effect of gene transfer.

Sl. 12: Inactivation of adenovirus using formaldehyde.

Sl. 13: Transfection of NIH3T3 cells in the presence of Moloney virus.

Sl. 14: Research on whether the effect of gene transfer in the transfection of NIH3T3 cells with transferrin-polylysine DNA complexes can be attributed to Moloney virus.

Sl. 15: Interaction between transferrin and receptors play a role in the effect of Moloney virus gene transfer.

Sl. 16: Effect of pH on the gene transfer effect of retroviruses.

Sl. 17: Influenza-hemagglutinin peptide; liposome leakage experiment.

Sl. 18: Transfection of K562 cells in the presence of influenza peptide, polylysine pl6pL conjugate.

Sl. 19: Transfection of HeLa cells in the presence of influenza peptide-polylysine conjugate p16pL.

Sl. 20: Transfection of HeLa cells with transferrin-polysine conjugates in the presence of influenza peptide-polylysine conjugate p4IpL.

Sl. 21: In situ evidence of beta-galactosidase expression after transfection of HeLa cells in the presence of adenovirus.

Sl. 22: Transfection of cells with a 48 kb cosmid in the presence of adenovirus

A: HeLa cells

B: Neuroblastoma cell

Sl. 23: Preparation of adenovirus-polylysine conjugate by chemical coupling.

Sl. 24: Transfection of K562 cells using chemically linked adenovirus conjugates.

Sl. 25: Transfection of HeLa cells using chemically linked adenovirus conjugates.

Sl. 26: Binding of polylysine to adenovirus by transglutaminase.

Sl. 27: Transfection of mouse hepatocytes using transglutaminase-linked adenovirus conjugates.

Sl. 28: Increasing transfection efficiency with transglutaminase-linked adenovirus conjugates.

Sl. 29: Transfection of HeLa cells with biotin-streptavidin-linked adenovirus conjugates.

Sl. 30: Transfection of K562 cells with biotin-streptavidin bound adenovirus conjugates.

Sl. 31: Transfection of neuroblastoma cells with kb cosmid using biotin-streptavidin-linked adenovirus.

Sl. 32: Transfection of hepatocytes in the presence of chloroquine or in the presence of adenovirus.

Sl. 33: Transfection of K562 cells in the presence of various endosomes

Sl. 34: Comparison of transfection protocols at the cellular level with beta-galactisidase as a proof gene, in the presence of various endosomolytic agents.

Sl. 35: Persistence of luciferase expression in confluent, undifferentiated hepatocytes.

Sl. 36: Comparison of expression in HeLa cells transfected in the presence of chicken embryo lethal virus (CELO) in free form and with CELO virus attached to polylysine via biotin-streptavidin.

Sl. 37: Transfection of myoblasts and myotubes with DNA/transferrin-polylysine complexes in the presence of free adenoviruses and in the presence of biotin/streptavidin bound adenoviruses.

Sl. 38: Delivery of DNA to cultures of mouse primary myoblasts and myotubes.

Sl. 39: Comparative analysis of adenovirus D1312 and CELO virus in transfection of HeLa cells and C2C12 myoblasts.

Sl. 40: Enhancement of CELO myoblast virus delivery using lectin ligands.

Sl. 41: Expression of full-length factor VIII cDNA in C2C12 myoblast and myotube cultures.

Sl. 42: Increase in DNA delivery by adenovirus proteins.

A) HeLa cells

B) Fibroblasts

Sl. 43: Galactose-influenza peptide conjugates for DNA transfer into hepatocytes.

Sl. 44: Galactose-adenovirus conjugates for DNA transfer into hepatocytes.

Sl. 45: Transfer of DNA with transferrin-polylysine in the presence of rhinovirus

A) Free rhinovirus

B) conjugated rhinovirus

Sl. 46: Transfection of primary human melanoma cells with combined complexes containing adenovirus conjugates.

Sl. 47: Liposome yielding experiment of amphipathic peptides.

Sl. 48: Experiment on erythrocyte leakage of amphipathic peptides.

Sl. 49: Transfection of BNL CL.2 cells in the presence of amphipathic peptides.

Sl. 50: Transfection of NIH3T3 cells in the presence of amphipathic peptides.

Sl. 51: Expression of interferon alpha in HeLa cells transfected in the presence of various endosomolytic agents.

The aim of the present invention was to improve the transfer of nucleic acid into more eukaryotic cells. (The term "transfer" in the sense of this invention means, beyond the introduction of the nucleic acid complex into the cell through the cell membrane, the transport of the complex or nucleic acid released therefrom inside the cell, as long as does not reach the appropriate position to express itself). Higher eukaryotic cells are well known and do not include yeasts. See “Molecular Biology of the Gene, James D. Watson et al., Benjamin-Cummings Publishing Company, Inc., p. 676-677 (1987).

Most viruses achieve their entry into eukaryotic cells through mechanisms that basically correspond to the mechanism of receptor control of endocytosis. Viral infection, based on this mechanism, generally begins with the binding of virus particles to receptors on the cell membrane. After that, the virus enters the cell. This entry process follows the usual pathway, which corresponds to the entry of physiological ligands or macromolecules into the cell: receptors on the cell surface cluster and the membrane turns inward, forming a bubble surrounded by an interwoven coating.

After that, the bubble is released from the coating, acidification occurs inside it by means of a proton pump located in the membrane. This promotes the release of the virus from the endosome. Depending on whether the virus has a lipid coating or not, two types of virus release from the endosome come into consideration: in the case of t. the so-called "golic" viruses (for example adenovirus, poliovirus, rhinovirus) it was assumed that low pH causes changes in the configuration of viral proteins. This highlights the hydrophobic domains, which are not accessible at physiological pH. These domains therefore acquire the ability to interact with the endosomal membrane and thereby cause the release of the viral genome from the endosome and into the cytoplasm. As for viruses with a lipid coating (eg, vesicular stomatitis virus, Semliki Forest virus, influenza virus), it is assumed that low pH modifies the structure or configuration of some viral proteins, thereby promoting fusion of the virus membrane with the endosomal membrane. Viruses that penetrate the cell through this mechanism have certain molecular features that allow them to break the membrane of the endosome in order to enter the cytoplasm.

Other viruses, eg enveloped Sendai viruses, HIV and some Moloney leukemia viruses, or non-enveloped SV40 and polyoma viruses, do not need low pH to enter the cell. They can either lead to fusion with the membrane directly on the cell surface (Sendai virus, probably HIV) or they are capable of triggering mechanisms of tearing the cell membrane or passing through it. Viruses, which are pH independent, are also thought to be capable of being used by endocytosis (McClure et al., 1990).

When solving the problem of the invention, the initial premise was to use the mechanism of some viruses for entry into eukaryotic cells, in order to improve the transfer of nucleic acid complexes into the cell, thereby enhancing expression.

Attempts have been made to introduce proteins together with viruses into the cell (Otero and Carrasco, 1987). It was found that the permeability achieved in the cell by the virus was used to release macromolecules. These processes appeared to be fluid phase mechanisms.

Using epidermal growth factor. EGF, conjugated to a toxin, this natural ligand, which entered the cell by endocytosis after binding to its receptor, was found to enter the same endosome together with the adenovirus, which also entered the cell by receptor-driven endocytosis, and was released from that endosome. again together with the virus, into the cytosol (FitzGerald et al., 1983).

The present invention surprisingly found that certain agents (eg viruses, viral components or other compounds), which exhibit the characteristics of certain viruses with regard to their mechanism of entry into eukaryotic cells, significantly increase the expression of nucleic acid introduced into the cell as part of the complex. This discovery is particularly surprising, because the complexes of nucleic acids, accepted into the cell, are very large.

The present invention therefore relates to a composition for transfection of higher eukaryotic cells with a complex of nucleic acid and a substance having an affinity for nucleic acid, which substance can be bound to an internalizing factor characterized by containing an agent capable of being drawn into the cells to be transfected, either "per se" or as a component of the nucleic acid complex, or to release the contents of the endosomes in which the complex is located after entering the cell, into the cytoplasm.

This agent is further referred to as an "endosomolytic agent". The ability of endosomolytic agents to be accepted into cells and to release the contents of endosomes, in which they are located after entering the cell, into the cytoplasm, is referred to below as the "acceptance function". This function includes the ability to be actively taken into cells by receptor-dependent endocytosis, or passively, via the liquid phase or as a constituent of nucleic acid complexes, and the ability to disrupt endosomes, which is generally referred to as endosomolysis.

In one embodiment of the invention, the endosomolytic agent is a virus. In the second, the endosomolytic agent is a viral component. The virus or viral component used in these embodiments of the invention is referred to below as "free" virus (component).

In the field of the present invention, the effect of an increased dose of free adenovirus on the gene transfer capacity of a constant amount of transferrin-polylysine conjugate in HeLa cells was investigated, using the luciferase gene as a reference gene. The increase in gene transfer caused by the addition of free adenovirus reaches a maximum at 1x104 virus particles per cell, which is a number that corresponds to the approximate number of adenovirus receptors per HeLa cell. An up to 2000-fold increase in luciferase expression, compared to the expression achieved with transferrin-polylysine conjugates alone, corresponds to a higher dose of virus. In the second series of experiments, the capacity of limiting amounts of the conjugate-DNA complex was examined, in the presence of a constant dose of free adenovirus. Cellular uptake of adenovirus was found to increase transferrin-polylysin-directed gene transfer over a wide range of DNA doses. The maximum intensity of gene expression achieved using the conjugate-DNA complex corresponded to the intensity achieved with 100 times less DNA, when adenoviruses were used to increase transfection efficiency.

The effect of adenoviral infection on gene transfer was significant for uncomplexed DNA as well as for DNA complexed with polylysine or transferrin-polylysine conjugates (Fig. 3A). According to this analysis, adenoviral infection did not significantly increase the transfer of bare, uncomplexed DNA during transfection. In contrast, the transfer of DNA complexed with polylysine or transferrin-polylysine conjugates was increased by adenoviral infection. This effect, however, is significantly greater for transferrin-polylysine conjugates. Since the polycationic part of the conjugate molecule not only serves to attach transferrin to DNA, but also significantly affects structural changes in DNA (Compaction into toroidal structures, Wagner et al., 1991a), these experiments could not initially distinguish whether the observed effects on the basis of enhanced fluid-phase transport of polycationic condensed DNA or increased acceptance of the receptor-bound conjugate-DNA complex by the virus. To distinguish between these possibilities, further related experiments were performed (Fig. 3b). Association of transferrin-polylysine-DNA or polylysine-DNA complexes, at low temperature without internalization, allowed removal of excess complexes in the fluid phase prior to adenoviral infection (FitzGerald et al., 1983). When given in this form, delivery of receptor-bound transferrin-polylysine-DNA complexes was significantly increased by the addition of adenoviral particles, while this was not the case for polylysine-DNA complexes. Therefore, the entry of DNA via receptor management of endocytosis is particularly enhanced.

Furthermore, an analysis of the specific adenoviral function, which leads to enhanced receptor-driven gene transfer, was performed (Fig. 3C). Mild heat treatment of virions does not alter their ability to bind to target cell membranes, but does affect their capacity to disrupt endosomes after internalization (Defer et al., 1990). Therefore, the different effects of viral association and viral input can be evaluated separately. In this assay, heat inactivation of adenoviruses completely abolishes their ability to enhance receptor-directed gene transfer. This suggests that the capacity of adenoviruses to disrupt endosomes as part of their entry mechanism is what specifically affects the enhancement of gene release via the transferrin-polylysine conjugate. The fact that for recovery defective types of viruses can lead to an increase in gene expression, confirms the assumption that this phenomenon does not depend on the function of recovery, but on the function of virion acceptance.

In order to exclude the possibility that the enhancement of gene expression can be attributed to the possible transactivation of the introduced gene by the virus, experiments were performed with a cell line, which constitutively expresses the RSV-LTR luciferase gene: adenoviruses do not show effects on that cell line, while in the parent line, in which if the gene was introduced via a transferrin-polylysine conjugate, there is a significant increase in gene expression. This finding demonstrates that adenovirus affects pre-transcriptional events and that its enhancing effect on gene transfer therefore acts at the level of gene transfer rather than at the level of gene expression (Fig. 5).

Research was also carried out within the framework of the invention, in order to find out what effect free adenovirus has on gene transfer via transferrin-polylysine conjugates in selected cell lines. It was found that the presence of transferrin receptors on the target cells is necessary, but not sufficient in every case to enable gene transfer by transferrin-polylysine conjugates. The cell-specific factors on which the fate of endosome-internalized conjugate-DNA complexes depends are extremely important determinants of the levels of gene transfer that can be achieved by this pathway. With this in mind, selected cell lines were tested for gene transfer with transferrin-polysin conjugates as well as for increasing gene transfer with adenoviruses (Fig. 4). Cystic fibrosis cell line (CTF1) cells showed moderate levels of luciferase gene expression after treatment with transferrin-polylysine-DNA complexes. This level of expression was significantly increased by treatment with adenovirus d1312. On the contrary, KB cells treated with transferrin-polylysine-DNA complexes showed levels of luciferase gene expression barely above baseline, despite the presence of the transferrin receptor. Adenovirus d1312 treatment, however, resulted in clearly detectable luciferase activities in these cells. Adenovirus treatment of HeLa cells had a similar effect, although the effect was much stronger in these cells. Since HeLa cells and KB cells possess approximately the same number of receptors for adenovirus, the difference in the increase in gene transfer may reflect the number of transferrin receptors characteristic of each cell type. However, in contrast to these results, the WI-38 and MRC-5 cell lines, which are known to support adenoviral infection very poorly (Precious and Russell, 1985), showed very little increase with d1312 in gene expression achieved by the DNA conjugates alone. complex. Treatment with free virus, eg, adenovirus, appears to increase gene transfer by conjugate-DNA complexes in those cases where gene transfer is possible by receptor-directed endocytosis, as in the case of CFT1 cells, and in some examples where gene transfer by this route appears to be ineffective, as with HeLa and KB cells.

The level of enhancement achieved varies considerably among different target cells, suggesting that this effect is a function of the number of viral receptors, eg adenovirus receptors, of a cell type, as well as the number of transferrin receptors.

In the case of using a free virus, it is preferred to conjugate to the internalizing factor a substance having an affinity for nucleic acid, usually an organic polycation. According to the invention, however, it was found that under certain circumstances, in the presence of a free virus, only DNA complexes with a substance having an affinity for nucleic acid can be introduced into the cell, i.e. without an internalizing factor. It has also been found that some cell lines can introduce through the fluid phase complexes, which consist of a nucleic acid and a substance that has an affinity for the nucleic acid, if the concentration of the complex is high enough. Experiments carried out within the framework of the present invention and some earlier ones have shown that an essential element for the capacity to accept nucleic acid complexes is their compactness, which can be attributed to the condensation of nucleic acid with a substance having an affinity for nucleic acid. If the substance, which has an affinity for the nucleic acid, has sufficient binding capacity to the cell surface for entry into the cell together with the virus, and which is capable of making the complex almost electroneutral and condensing the nucleic acid into a complete structure, then there is no need to increase the entry capacity by covalently linking the internalizing factor to a substance that has an affinity for nucleic acid, for the purpose of transferring the complex into the cell by receptor-controlled endocytosis. Many cells have a relatively high affinity for some substances that have affinities for nucleic acid, so that nucleic acid conjugates and binding factor are taken into the cell without the need for internalizing factor. This applies, for example, to hepatocytes, which in the context of the present invention have been found to accept DNA-polylysine complexes.

In the presented embodiment of the invention, the endosomolytic agent is a virus that is bound to a substance, that has an affinity for nucleic acid and that has the ability to enter the cell as part of the conjugate/nucleic acid complex and to release the contents of the endosomes, in which the complex is located after entering the cell , in the cytoplasm.

In another preferred embodiment, the endosomolytic agent is a viral component that is attached to a substance that has an affinity for nucleic acid and that has the ability to enter the cell as part of a conjugate/nucleic acid complex and to release the contents of the endosomes in which the complex is located after entering the cell. , into the cytoplasm.

Viruses or viral components linked to a nucleic acid domain, regardless of the type of linkage, are referred to below as "viral conjugates".

Viral conjugates, which are also the subject of the present invention, contain a virus or a viral component as an integral part of their functional structure and combine the advantages of vector systems, which are based on internalizing factor conjugates, with the advantages that viruses bring to these systems.

Furthermore, viral conjugates, according to these embodiments of the invention, prevent the basic limitation inherent in known systems of molecular conjugates, in that unlike known conjugates, used for gene transfer via receptor-driven endocytosis, they have a specific mechanism that allows them to be released from the system cell cavities.

A vector system using viral conjugates represents a basic conceptual departure from recombinant viral vectors in that the foreign DNA to be transported is carried on the outside of the virion. Therefore, the viral conjugates according to the presented embodiment of the invention can transport very large gene constructs into the cell, without limitation.

The suitability of a virus, to be used as a free or bound virus or part of a virus as a viral component, within the scope of the present invention is determined by its acceptance factor, as defined herein. Suitable viruses are, on the one hand, those that have the ability to enter the cell by receptor-driven endocytosis during transfection of the cells with the nucleic acid complex and that contribute to their release - and therefore the release of the nucleic acid - from the endosomes into the cytoplasm. Such viruses are those discovered here, as well as other viruses capable of being accepted by an individual cell, causing the release of endosomal contents into the cytoplasm.

For examples of viruses and higher eukaryotic cells they can penetrate, there are references from Fields B.N. and Knipe, D.M. (1990). The susceptibility of a given cell line to transformation by a virus as a facilitator of conjugate entry as “free virus” is dependent on the presence and number of target cell surface receptors for the virus. With respect to the adenoviral cell surface receptor, the methods of Svensson, 1990, and Defer, 1990 were proposed for determining its number on HeLa and KB cells. It was thought that the adenovirus receptor was rather widely expressed.

Suitable viruses include, on the one hand, those capable of entering the cell via receptor-directed endocytosis during transfection of the cells with the nucleic acid complex and which contribute to their release, and thus the release of the nucleic acid, from the endosomes into the cytoplasm. Without wishing to be bound by that theory, this activity of endosomolysis can, in the case of using a free virus, be used to transfer nucleic acid complexes into the cell, insofar as these complexes are transferred together with the virus from the endosomes to the cytoplasm, assuming that they arrive in the same endosomes. as well as viruses that are internalized. When the complexes contain the virus in bound form, they are used by the viral endosomolytic activity and transported from the endosomes to the cytoplasm. This prevents the fusion between endosomes and lysosomes, thus also the enzymatic degradation that normally occurs in these cell organelles.

In particular, viruses, which are capable of composition according to the invention and whose acceptance function, appearing at the beginning of infection, is observed by receptor-driven endocytosis, include on the one hand viruses without a lipid coating, such as adenovirus, poliovirus, rhinovirus, and on the other hand coated viruses, vesicular stomatitis virus, Semliki Forest virus, influenza virus; pH-dependent Maloney virus types are also suitable. Viruses that can be particularly used in the practice of the invention include Adenovirus subgroup C, type 5, Semliki Forest virus, vesicular stomatitis virus, poliovirus, rhinoviruses and Maloney leukemia virus.

The use of a DNA virus that does not have reverse transcriptase has the advantage in the present invention that transfection in the presence of such a virus does not lead to the creation of viral DNA in the transfected cell.

It has been shown in the present invention that Rhinovirus HRV2, a representative of the Picorna virus group, enhances the expression of the reference gene. The effectiveness of Rhinovirus was expressed both in free form and in the form of viral conjugates.

Within the scope of the present invention, the indicated viruses, assuming that they are accepted into the cell and that they release the contents of the endosomes in which they have arrived, include, in addition to wild-type mutants that have lost certain functions of the wild type, which differ from their function of acceptance, in particular the ability to are restored, as a result of one or more mutations.

Mutants are produced by common mutagenesis processes, mutations in virus-protein regions, which are responsible for repair functions and which can be complemented by an envelope sequence. These mutants include, for example in the case of adenoviruses, ts-mutants (temperature-sensitive mutants), E1A- and E1B-mutants, mutants exhibiting mutations in MLP-driven genes (Berkner, 1988) and mutants exhibiting mutations in regions of some capsid proteins. Viral species that have the appropriate natural mutations are also suitable. The ability of viruses to renew themselves can be tested, for example, using "plaque" experiments known from the literature, in which cell cultures are covered with suspensions of different concentrations of virus, and the number of destroyed cells, which are visible through the coating (plaque) is recorded (Dulbecco, 1980).

Other viruses that may be suitable for use within the scope of the invention include the so-called defective viruses, i.e. viruses that lack the function required for autonomous viral renewal in one or more genes, for which they require helper viruses. Examples of this category are DI-particles (Defective Interfering Particles) which are derived from the infectious standard virus, have the same structural proteins as the standard virus, have mutations and require the standard virus as a helper virus for recovery (Huang, 1987; Holland, 1990). Examples of this group also include satellite viruses (Holland, 1990). Another group of parvovirus classes is called adeno-associated virus (Berns, K.I. 1990).

Since the entry cycles of many viruses have not been fully characterized, it is possible that there are other viruses that will exhibit the endosomolytic activity necessary for their usefulness in the present invention.

In addition, live attenuated vaccines (Ginsberg, 1980) or vaccine strains may be suitable within the scope of this invention.

Viruses mentioned within the scope of the present invention also include inactivated viruses, eg viruses inactivated by chemical treatment, such as treatment with formaldehyde, UV. radiation, chemical treatment combined with UV radiation, eg psoralen/UV radiation, gamma radiation or neutron bombardment. Inactivated viruses, such as those used for vaccines, can be prepared by standard methods known from the literature (Davis and Dulbecco, 1980; Hearst and Thiry, 1977) and tested for their ability to increase transfer of DNA complexes. In experiments performed within the scope of the present invention, adenovirus preparations were inactivated using a conventional UV sterilization lamp or formaldehyde. Surprisingly, it was found that the degree of virus inactivation was significantly greater than the reduction in gene transfer effect, which was achieved when adenovirus was added to the transfection medium.

Experiments conducted by the inventors with preparations of biotinylated adenovirus inactivated by the psoralen/UV-irradiation combination, which was coupled to polylysine-bound streptavidin, also showed that as a result of inactivation, the virus titer was reduced significantly more than the gene transfer capacity. This is a clear indication that the mechanisms associated with normal renewal in an active virus can be disrupted without the disrupted effect being essential for gene transfer.

The term "viral components" refers to parts of the virus, e.g. the protein part released from the nucleic acid (empty viral capsid which can be produced by recombinant methods, e.g. Ansardi et al., 1991; Urakawa et al., 1989), proteins obtained by fractionation or peptides which have the endosomolytic function of the intact virus. These viral components can be produced synthetically, depending on their size, either by peptide synthesis or recombinant methods. In the present invention, it has been shown that adenovirus proteins conjugated via biotin/streptavidin to polylysine, increase gene transfer. Examples of fragments or proteins from viruses other than adenovirus that are essential for internalization include influenza virus hemagglutinin (HA). The N-terminal sequence of influenza virus hemagglutinin, the HA2 subunit, is responsible for releasing the virus from endosomes. It has been shown that peptides of this sequence, consisting of 20 amino acids, can fuse lipid membranes and partially open or destroy them (Wharton et al., 1988). In the present invention, authentic and modified influenza peptides have been successfully used in various embodiments. Other examples are protein coats of retroviruses, eg HIV gp41 (Rafalski et al., 1990) or parts of these viral proteins.

The use of viruses that are themselves capable of entering a cell and thus act as internalization factors is only one aspect of the present invention.

Viruses or viral components that do not themselves bring the capacity to bind to and enter the cell are instead used as viral conjugates, as noted above. Binding to a DNA splicing domain, eg a polycation, ensures that the virus (component) acquires a high affinity for DNA molecules and therefore complexes with it and is transported into the cell as a component of a nucleic acid complex, which also contains a conjugate of the internalization factor and the DNA binding domain. In addition to the transmission effect achieved in this way, binding of the virus (component) to the nucleic acid binding domain may also result in an improvement of its endosomolytic properties.

By selecting the other internalization factors described herein, virtually any higher eukaryotic cell can be transfected with the compositions of the present invention.

A simple test can determine whether a virus (component) has an acceptance function and whether it can be used in the practice of the invention. In this experiment, for example to check the applicability of the virus as a free virus, the target cells are brought into contact with the DNA complex in the presence or absence of the virus. The amount of DNA complex released into the cytoplasm can then be easily determined by detecting the product of a marker gene, eg, luciferase. If the presence of virus causes the DNA complex to be taken up and released into the cytoplasm at a higher level than in the absence of virus, this can be attributed to the uptake function of the virus. It is also possible to compare the level of uptake of the DNA complex with the test virus compared to another virus known to have a favorable uptake function, eg adenovirus subgroup C, type 5. Tests of this type can also be applied to viral conjugates, additional parameters such as different internalization factor conjugates in different amounts can be subjected to such tests. In addition, a person trained for this work can easily perform an experiment of this type, if desired in combination with other tests such as, for example, liposome leakage experiments, to test virus components or other agents with potential endosomolytic activity for their ability to increase gene expression.

When intact viruses are used, tests are performed, preferably in parallel with preliminary tests of the ability of the virus to enhance transmission, to see if the virus is capable of recovery. Studies on the ability to renew are carried out using the "plaque" test (see above) or the CPE test or by determining earlier gene expression in the case of cytopathic viruses or in the case of viruses that significantly interfere with the growth of host cells. For other viruses, methods specific to the virus in question are used, eg the hemagglutination test, or chemical-physical methods, eg the use of an electron microscope.

Within the scope of this invention, the best viruses, especially those used as free viruses, are those that can be prepared with a high titer, that are stable, that have low pathogenicity in their natural state and in which targeted elimination of the ability to renew is possible, especially adenoviruses. If a specific cell population is to be transfected, viruses that specifically infect that cell population can be used. If different cell types are intended to be targeted by transfection, viruses that are infectious for a wide range of cell types can be used.

The requirements for free virus compositions consist essentially in the fact that the virus preparation must be as pure as possible and that the stabilization buffer must be compatible with the individual virus.

In any case, only those viruses or viral components whose safety risks are as minimal as possible can be used for the therapeutic use of the invention, especially the risk of viral replication in the target cell and recombination of viral DNA with host DNA.

The entry mechanism of viruses that infect animal beings other than humans can usefully be used to enhance the uptake and release of DNA into higher eukaryotic cells, particularly human, as long as the virus expresses endosomal destruction activity in higher eukaryotic cells. Members of the adenovirus family have been isolated from avian species, amphibians, and various other animals.

See, for example, Laver, W.G. et al., 1971; Bragg, R.R. et al., 1991; Akopian, T.A. et al., 1991; Takase, K. et al., 1990; Khang, C. and Nagaraji, K.V., 1989; and Reece, R.L. et al., 1987. Amphibian, avian, bovine, dog, mouse, sheep, pig, and monkey adenoviruses, as well as human adenoviruses, can be obtained from the American Type Culture Collection, Rockville, Maryland (See Animal Virus Culture Collection Catalog, Antisera, Chlamydia , Rickettsia, Sixth Edition, 1990, C. Buck and G. Paulino Publishers, pp. 1-17).

One possible advantage of using a virus, e.g. adenovirus, from a different species could be reduced toxicity in target cells (e.g. chicken or frog adenovirus should not replicate or induce early gene expression in mammalian cells), reduced risk for the researcher preparing further adenovirus, compared to human adenovirus, and reduced binding to the target organism by antibodies against human or murine adenovirus. The absence of interference by human or mouse antibodies is particularly important when viruses are used in gene therapy in humans and mice.

Chicken adenovirus CELO (Chick Embryo Lethal Orphan Virus) does not show reactivity to antibodies that allow epitopes of most groups of adenoviruses that infect mammalian cells. In addition, CELO can be grown on egg embryos to reach high virus levels (0.5 mg/egg; Lavel et al., 1971). As shown in the Examples, CELO-polylysine conjugates enhance DNA delivery into HeLa cells at levels comparable to human adenovirus d1312. Therefore, the use of CELO conjugates to enhance DNA release offers great prospects in human gene therapy experiments.

Viruses of separate species are preferably used as constituents of viral conjugates in combinatorial complexes, as described herein.

In the virus-containing conjugates of the invention, the binding of the virus to the nucleic acid binding domain can be covalent or non-covalent, eg, the ionic bond in the case of a virus has regions on its surface proteins, which are acidic and therefore can bind to a polycation.

In the experiments of the present invention, complexes were formed under conditions that allow ionic interaction between adenovirus and polylysine, prior to complexation with DNA. Control experiments were performed under conditions where polylysine was first neutralized with DNA and therefore not free to bind adenovirus. In these experiments, complexes with ionically bound adenovirus were more successful.

Examples of viral components in the endosomolytic conjugates of the invention are empty viral capsids or viral peptides. Binding of the viral component to the linker nucleic acid domain can be covalent, eg by chemical coupling of the viral peptide to a polylysine, or non-covalent, eg ionic, in case the viral component has acidic residues to bind to the polycation.

The relationship of a virus or a viral component to a substance that has an affinity for nucleic acid can vary. In the case of influenza hemagglutinin peptide-polylysine conjugates, it was found in the present invention that gene transfer can be significantly enhanced when the viral peptide content in the conjugates is higher.

On the other hand, the present invention relates to methods of preparing viral conjugates, according to the invention.

Conjugates of viruses or viral components and substances that have an affinity for nucleic acid can be prepared (as internalizing factor-polycation conjugates) by connecting compounds or, if the viral component and the nucleic acid binding domain are polypeptides, by a recombinant method. With regard to preparation methods, reference is made to the disclosure EP 388 758.

Binding of viruses or viral proteins, i.e. peptides with polyamine compounds using a chemical method, can be carried out in a way that is already known for joining peptides, and if necessary, individual components can be supplied with connecting substances before the joining reaction (this measure is necessary if there are no functional groups which are suitable for coupling, e.g. mercapto or alcohol groups). Binding substances are bifunctional compounds that react first with the functional groups of individual components, after which the modified individual components are joined.

The connection can be done using:

a) Disulfide bridges, which can be re-cleaved under limited conditions (eg when succinimidyl-pyridyldithiopropionate is used (Jung et al., 1981).

b) Compounds that are substantially stable under biological conditions (eg thioethers by reaction of the meimido linker with the sulfhydryl groups of the linker attached to another component).

c) Bridges that are unstable under biological conditions, eg ester bonds or acetal or ketal bonds, which are unstable under slightly acidic conditions.

In experiments performed within the framework of the present invention, endosomolytic HA2-peptides of influenza-hemagglutinin were combined with polylysis, a chemical method using succinimidylpyridyldithiopropionate (SPDP). It has been shown that the modification of the peptide with polylysine increases the endosomolytic activity. Transfection experiments showed that the efficiency of transferrin-polylysine controlled gene transfer significantly increases if influenza-polylysine peptide conjugates are present together with transferrin-polylysine in the DNA complex.

Furthermore, within the scope of the present invention, the adenovirus was attached to the polylysine by a number of different methods. One method of conjugating virus with polylysine was carried out in a manner similar to the production of transferrin-polylysine conjugates (Wagner et al., 1990) after modification of damaged adenovirus d1312 using a heterobifunctional reagent. Unbound polylysine was removed by centrifugation. The binding capacity of DNA was demonstrated in a binding experiment using radiolabeled DNA.

(In K562 cells, in the absence of chloroquine, significantly higher gene transfer was found with complexes consisting of DNA, adenovirus-polylysine and transferrin-polylysine, than with unmodified adenovirus that is not bound to DNA. It was also found that significant gene expression occurs with only 0.0003µg DNA in 5x105 HeLa cells using polylysine-modified adenovirus

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

Another suitable method of preparing the viral conjugates of this invention is the method by means of enzymatic coupling of the virus or viral component with a substance having an affinity for nucleic acid, especially with polyamine, by means of transglutaminase.

The category of transglutaminase includes a number of different enzymes, which occur among others in the epidermis (epidermal transglutaminase), in the blood (factor XIII) and in the cells of various tissues (eg liver transglutaminase) (Folk, 1985). Transglutaminases catalyze the formation of ε-(γ-glutamyl)lysine bonds in the presence of Ca++ and with NH3 cleavage. The condition for this is that the appropriate glutamines and lysines, capable of reacting with the enzyme, must be present in the proteins.

Regardless of the ε-amino group of lysine, (poly)amines, such as ethanolamine, putrescine, spermine, or spermidine, can also be used as a substrate (Clarke et al., 1959). For now, it is not yet clear what are the critical factors that determine whether glutamine or lysine of a protein or polyamine can react with the enzyme. It is only known that polyamines can be bound by transglutaminase to numerous cellular proteins, such as cytokeratins (Zatloukal et al., 1989), tubulin, cell membrane proteins and influenza virus surface proteins (Iwanij, 1977).

Within the scope of the present invention, it has been shown that polylysine can be joined to adenoviruses by means of transglutaminase. It was found that coupling can be performed in the presence of glycerol. This has the advantage that a viral preparation, eg an adenovirus preparation containing glycerol as a stabilizer in the buffer, can be used directly for conjugation. By using adenovirus-polylysine conjugates that are complexed with plasmid-DNA together with transferrin-polylysine conjugates, it was possible to achieve many times higher gene expression than with transferrinpolylysine conjugates in the presence of adenovirus not combined with polylysine.

Another method of preparation of the conjugate, which is advantageous within the scope of the invention, consists in connecting the virus or viral component with a polycation via a biotin-protein bridge, especially a biotin-streptavidin bridge.

The known strong binding of biotin to streptavidin or avidin (Wilchek et al., 1988) was used to link adenovirus to polylysine, by modifying adenovirus with biotin and chemically conjugating streptavidin to polylysine, in a similar way to producing transferrin-polylysine conjugates (Wagner et al. ., 1990). Complexes consisting of DNA and streptavidin-polylysine, to which biotin-modified virus is bound, and optionally non-covalently bound polylysine, have a very high transfection efficiency even with lower concentrations of DNA. Particularly effective complexes are created when the biotin-modified virus first binds to streptavidin-polylysine, and binding to DNA occurs only in the second phase.

If desired, binding to biotin can also be performed using avidin.

It is also possible to establish a link between the virus (component) and the polylysine by biotinylating the virus, on the one hand, and conjugating the antibiotin antibody to the polylysine, on the other hand, and establishing the link between the virus and the polylysine by means of a biotin-antibody link, using standard commercially available polyclonal or monoclonal anti-biotin antibodies.

Conjugation between virus and polysin can also be achieved by conjugating the polysin to a lectin having an affinity for the virus surface glycoprotein, provided that the binding into such a conjugate is accomplished by means of a link between the lectin and the glycoprotein. If the virus itself does not have a suitable carbohydrate side chain, it can be suitably modified. The virus can also be attached to a substance, which has an affinity for nucleic acid, so that it is first modified on the surface with an antigen foreign to the virus (eg with digoxigenin DIG, which can be obtained from Boehringer Mannheim; or with biotin) and then the bond is established between the modified virus and a substance that has an affinity for nucleic xylin, through an antibody that binds to that antigen. Which particular method will be used to produce the conjugate according to the invention depends on various criteria.

For example, coupling using biotin is the least specific and therefore the most widely used method, while biotin-directed coupling creates a very strong non-covalent bond. The enzymatic reaction with transglutaminase has the advantage that it can be performed on a very small scale. Chemical coupling is generally used when larger amounts of conjugates need to be synthesized and this method is generally best when joining viral proteins or peptides. If inactivated viruses are used, inactivation is generally performed prior to conjugation, assuming that inactivation has not affected conjugation.

If a virus, eg adenovirus or its endosomolytic component, has an accessible binding domain, eg an acidic polycation binding domain, binding of the virus to the polycation may also be ionic. In this case, the positive charges of the polycation, which can be conjugated with the internalizing factor, are partially neutralized by the acidic domain of the virus (component), and the rest of the positive charges will mostly be neutralized by the nucleic acid.

If the substance that has an affinity for nucleic acid is an intermediate substance, it is modified with a binding agent that is suitable for individual joining of the virus (component), e.g. for joining with transglutaminase, it is modified with spermine or with a bifunctional group responsible for chemical coupling, eg with an active ester.

The relationship of the virus (component) to the nucleic acid binding substances can vary, which is usually determined empirically, eg by combining a constant amount of virus (component) with different amounts of polylysine and selecting the optimal conjugate for transfection.

In another embodiment of the invention, the viral component, eg an endosomolytic viral peptide can be modified for the purpose of binding directly to DNA. Finally, the peptide itself may contain a DNA binding domain, which can be achieved by producing the peptide using peptide synthesis and obtaining a good proportion of positively charged amino acids, particularly by extending the peptide preferably at the C-terminus.

In yet another embodiment of the invention, the endosomolytic agent is a non-viral, usually synthetic peptide. A peptide of this type is usually contained in the composition according to the invention in such a way that it is ionically bound to a substance with affinity for nucleic acid, eg to polylysine in the case of the DNA-internalizing factor-polylysine complex. Therefore, incorporation of the endosomolytic peptide into nucleic acid complexes is performed by linking the peptide via its acidic amino acid residues to a positively charged nucleic acid binding domain, preferably polysine.

Depending on the chemical structure of the peptide, particularly with respect to its terminal group, linking to polylysine can be accomplished by the methods described herein for linking peptides to polylysine. Finally, if a naturally occurring peptide is used, it can be modified with a suitable terminal amino acid as a coupling agent.

Another way is to incorporate non-viral endosomolytic peptides into nucleic acid complexes, to provide them with extensions that bind to DNA. The location of such extension must be such that it does not interfere with the endosomolytic activity of the peptide. Therefore, for example, peptides whose N-terminus is responsible for this activity are spread across the DNA linking sequences to the C-terminus. Extensions of this type can be homologous or heterologous cationic oligopeptides, e.g. single-oligolysine tail, or a natural DNA binding domain, e.g. a histone-derived peptide. Mostly these DNA binding sequences, as an integral part of the endosomolytic peptide, contain approximately 10 to 40 amino acids. This embodiment of the invention provides the possibility of a higher ratio of the endosomolytic sequence to the DNA binding sequence, than in the case of conjugates containing larger amounts of polycations in order to achieve a higher efficiency of the complex.

Non-viral endosomolytic peptides must fulfill the following requirements:

Given the endosomolytic activity, the permeability of the lipid membrane achieved by the peptide must generally be higher at low pH (5-6) than at pH 7. Furthermore, the disrupted membrane surfaces must be large enough to allow the passage of large DNA complexes (small pores are not sufficient). . In order to determine whether the peptide meets these requirements, in vitro tests can be performed using the peptide in free or bound form or embedded in a DNA complex. Such experiments may include liposomal permeation experiments, erythrocyte permeation experiments, and cell culture experiments to determine increases in gene expression. Tests of this type are described in the Experiments and Examples chapter. The optimal amount of peptide can be determined by preliminary titrations using experiments on the efficiency of the resulting gene transfer. It should be kept in mind that the effectiveness of different peptides and the optimal composition of the complex may depend on the cell type.

Peptides that destroy the membrane contain, mainly, amphipathic sequences, namely the hydrophobic side that reacts with the lipid membrane, the hydrophilic side that stabilizes the aqueous phase at the site of membrane rupture.

There are a number of examples in nature of membrane-disrupting peptides, usually small peptides or peptide domains of large polypeptides. Such peptides can be classified according to their natural function, namely, either membrane-disrupting peptides (eg, naked virus peptides) and/or membrane-binding peptides (eg, enveloped viruses). For the purpose of disrupting endosomes, in the context of synthetic peptides, both classes of peptide arrays can be useful. Most natural peptides are capable of forming amphipathic α-helices.

pH-specificity can be achieved by incorporating acidic residues on the hydrophilic side of the putative amphipathic α-helix, such that the helix can only form at acidic pH, but not at neutral pH, where the repulsion between negatively charged acid residues prevents helix formation. This property is also found in naturally occurring strings. (eg influenza HA-2 N-terminus).

A fully synthetic, rationally labeled, peptide (amphipathic) with specific pH-dependent membrane disruption properties has been described (Subbarao et al., 1987; Parente et al., 1990). It was shown that this peptide (in its free form) creates only small pores in the membranes, and allows the passage of small compounds (Parente et al. 1990).

According to the embodiment of the invention in which non-viral, possibly synthetic peptides are used, the following stages are usually carried out: a series of amphipathic peptides is selected from those occurring in nature or from the group of artificial peptides. An overview of examples of peptides of this type, known in practice, is given in table 2.

If necessary, acid residues (Glu, Asp) are introduced to make the activity of destruction of the peptide membrane as specific as possible, considering the pH (eg double acid mutant hemagglutinin of the influenza peptide, according to example 35, marked with p50).

If necessary, acidic residues can also be introduced in order to facilitate the binding of the peptide to the polysine. One way to obtain such a polycationic binding domain can be the introduction of an (acidic) extension, eg an oligo-Glu-extension.

Endosomolytic peptides suitable for the present invention can also be obtained by fusing naturally occurring sequences with artificial ones. In the present invention, experiments were carried out with different peptides that were derived from the synthetic peptide GALA, described by Parente et al., 1990. Some of the derivatives used in the experiments of the present invention were obtained by combining the Gala peptide or its modifications, with sequences of influenza peptides or its modifications, for example peptides labeled with EALA-Inf and EALA-P50, according to example 33.

The length of the peptide sequence may be critical with respect to the stability of the amphipathic helix; increasing the stability of short domains derived from natural proteins can be achieved by lengthening the helix.

In order to increase the endosomolytic activity of the peptide, homodimers, heterodimers or oligomers can be formed. In experiments of the present invention, the P50 dimer has been shown to have much greater activity than the monomer.

The present inventors have demonstrated the effect of synthetic peptides on DNA uptake mediated by transferrin-polylysine conjugates. A number of different peptides were synthesized, their permeability to liposomes and erythrocytes was tested, and their influence on the expression of luciferase in TIB cells and in NIH 3T3 cells was tested. In another embodiment of the invention, the endosomolytic agent may be a non-peptide amphipathic substance. The requirements that such a substance must meet to be suitable for the present invention are essentially the same as for amphipathic peptides, i.e. the ability to be incorporated into nucleic acid complexes, pH specificity, etc.

From another aspect, the invention relates to complexes that are accepted into higher eukaryotic cells, which contain nucleic acid and a conjugate capable of forming a complex with nucleic acid, for the introduction of nucleic acid into higher eukaryotic cells.

The complexes are characterized in that the conjugate consists of a substance having an affinity for nucleic acid and an endosomolytic agent that is bound to a substance having an affinity for nucleic acid and is capable of being internalized into the cell as a conjugate/nucleic acid complex and releasing the contents of the endosome. in which the complex is located after entering the cell, in the cytoplasm.

Nucleic acid complexes, used within the scope of the invention, are mainly those in which nucleic acid is complexed with a substance that has an affinity for nucleic acid, in such a way that the complexes are substantially electroneutral.

In a favorable embodiment of the invention, the endosomotic agent is a virus or a viral component covalently bound to a polycation.

Within the scope of the present invention, endosomolytic conjugates include, in addition to conjugates in which endosomolytic agents are ionically bound to the binding domain of DNA, and endosomolytic agents that bind to DNA directly, e.g., via their basic extension, although "conjugates" of this type, in fact, they do not get by conjugation, i.e. by connecting two components to each other. The function of endosomolytic agents of this type as compounds of the composition according to the invention is independent of whether they are synthesized by the conjugate of the endosomolytic agent and the DNA binding domain or whether the DNA binding domain was originally present in the endosomolytic agent.

In another advantageous embodiment of the invention, the complexes contain, in addition to the endosomolytic conjugate, another conjugate in which the substance, which has an affinity for the nucleic acid, in the case of an endosomolytic polycationic conjugate generally of the same cation as in the conjugate, is conjugated to an internalizing factor that has an affinity for the target cell. This embodiment of the invention is particularly useful when the target cell has no or few receptors for the virus used as part of the endosomolytic conjugate. Another application of this embodiment of the invention is when a viral component is used, for example a peptide that comes in nature, as well as a modified peptide, non-viral, can also be a synthetic endosomolytic peptide or a virus from other species, which do not have the ability to penetrate cells by themselves which needs to be transfected. In the presence of an additional factor conjugate that internalizes the binding factor, endosomolytic conjugates benefit from the internalizing ability of the second conjugate by being complexed to nucleic acid together with the second conjugate and being taken up into the cell as part of the resulting complex, hereafter referred to as " combination complex" or as a "triple complex". Regardless of this theory, combination complexes are taken up into cells, either by binding to a surface receptor that is specific for an additional internalizing factor, or, for example, in the case of a virus or viral component, binding to a viral receptor or binding to both receptors, internalization by receptor-driven endocytosis . When the endosomolytic cores are released from the endosomes, the DNA contained in the complexes is also released into the cytoplasm, thus avoiding lysosomal degradation.

In experiments of the present invention, almost all HeLa cells can be transfected with free adenovirus. The effectiveness for hepatocytes can be further improved if triple DNA complexes are used in which the reference DNA is complexed with polylysine-transferrin conjugates and linked to the adenovirus. Here, localization of the endosomolytic virus and ligand-receptor complex in the endosome is guaranteed, bringing transfection into almost all cells for a range of cells, such as BNL.CL2 and HepG2 cells. In this case, both viral and transferrin receptors on the cell surface can act to capture triple DNA complexes. However, it can also be predicted that DNA ternary complexes can be internalized only by the action of cellular ligand/receptor association. Such a situation can be approximated in experiments where triple DNA complexes, containing transferrin, gain access to K562 cells mainly through the transferrin receptor, rather than through the adenovirus receptor.

Unexpectedly, triple complexes transferred DNA even when present for DNA transfer at very low levels. Thus, with the addition of 30 pg DNA/3x105 cells, 1.8x104 units are obtained (resulting from the luciferase-encoded plasmid). At that entrance, there are only 60 DNA molecules and 1 PFU of virus per cell. This should be compared with the less efficient cation deposition process, which uses 105 DNA molecules per cell (Molecular Cloning, Samobrook, J. et al. 2nd edition, Vol. 3, pp. 16.39-16.40, 1989). Therefore, the present invention represents a significant advance in practice, since it enables effective transformation of higher eukaryotic cells with a very small amount of DNA.

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

1) Broader possibility of applying gene transfer technology with nucleic acid complexes, since the endosomolytic agent itself, especially in the case when a virus (component) is used, may contain an internalizing factor or may also be complexed with DNA together with another internalizing factor (e.g. transferrin or asialofetuin, etc.). In this way, it is possible to use the positive effect of the virus even for cells that do not have any receptor for the virus in question.

2) Improving the efficiency of gene transfer, since the binding of endosomolytic conjugates to DNA ensures that they are co-uptake into cells. Coordinated uptake and release of virus and DNA also increases the possibility of reducing the amount of DNA and virus required for efficient gene transfer, which is of particular importance for in vivo use.

The term "internalizing factor" refers, for the purposes of the present invention, to ligands or their fragments, which after binding to the cell are internalized by endocytosis, namely by receptor-controlled endocytosis, or to factors whose binding or internalization is performed by binding to elements of the cell membrane.

Suitable internalizing factors include ligands such as transferrin (Klausner, R.D. et al., 1983), conalbumin (Sennett, C. et al., 1981), asialoglycoproteins (such as asialotransferrin, asialorosomucoid or asialofetuin) (Ashwell, G. et al. , 1982), lectins (Goldestein et al., 1980, Shardon, 1987) or substances containing galactose, which are internalized by the asialoglycoprotein receptor, such as: mannosylated glycoproteins (Stahl, P.D. et al., 1987), lysosomal enzymes (Sly, W. et al., 1982), LDL (Goldstein, J.L. et al., 1982), modified LDL (Goldstein, J.L. et al., 1979), lipoproteins that are taken up into the cell via receptors (apo B100/LDL); viral proteins such as the HIV protein gp120; antibodies (Mellman, L.S. et al., 1984, Kuhn, L.C. et al., 1982, Abrahamson, D.R., et al., 1982) or fragments thereof against cell surface antigens, e.g., anti-CD4, anti -CD7; cytokines such as interleukin-1 (Mizel, S.B. et al., 1987), interleukin-2 (Smith, K.A. et al., 1985), TNF (imamure, K. et al., 1987), interferon (Anferson, P . et al., 1982), colony stimulating factor (Walker, F. et al., 1987); factors and growth factors such as insulin Marshall, S., 1985), EGF (Carpenter, G., 1984), platelet-derived growth factor (Heldin, C.H. et al., 1982), transforming growth factor β (Massague, J . et al., 1987), insulin-like growth factor I (Schalch, D.S. et al., 1986), LH, FSH, (Ascoli, M. et al., 1978), growth hormones (Hizuka, N. et al. , 1981), prolactin (Posner, B.I. et al., 1982), glucagon (Asada-Kubota, M. et al., 1983), thyroid hormones (Cheng, S. - Y. et al., 1980); α-2-macroglobulin proteases (Kaplan, J. et al., 1979); and "disarmed" toxins. Further examples are immunoglobulins or their fragments as ligands for the Fc receptor or anti-immunoglobulin antibodies, which bind to Sigs (surface immunoglobulins). Ligands can be of natural or synthetic origin. See: Trends Pharmacol. Sci. 10:458-462 (1989) and references cited therein.

The essential requirements for the convenience of such factors, according to the present invention, are as follows,

a) they can be internalized by the specific type of cell in which the nucleic acid must be introduced, and their ability to be internalized is not affected, or only slightly affected, if they are conjugated with a binding factor and

b) within this property, they are capable of carrying nucleic acid "on their back" into the cell.

In the experiments performed according to the invention, a wide range of use of the invention was demonstrated with respect to the internalizing factor, or to the additional internalizing factor, i.e. in combination complexes, using human and mouse transferrin-polylysine conjugates, asialofetuin-pL conjugates, galactose-pL conjugates, wheat agglutinin conjugates germ, by T-cell-specific gp120-pL and antiCD7-pL conjugates and by DNA polylysine conjugates that do not contain any internalizing factor. In addition, the properties of viral conjugates, according to the invention, were demonstrated using complexes of DNA and polylysine conjugated virus (or viral component) that did not contain additional conjugates of the internalizing factor with the binding factor

Specific preliminary tests can be performed to determine whether, in the event that the endosomolytic agent is a free virus, or in the event that the endosomolytic agent is a virus or a viral compound or a non-viral peptide that is part of an endosomolytic conjugate, the use of an internalizing factor facilitates or enhances the uptake of the nucleic acid complex. acid. These tests include parallel transfections with nucleic acid complexes, primarily without an (additional) internalization factor, e.g. in the case of viral conjugates with complexes consisting of a nucleic acid and a viral conjugate, and secondarily with complexes in which the nucleic acid is complexed with another conjugate which consists of an additional internalization factor, for which target cells have a receptor, and a substance that has an affinity for nucleic acid.

If an internalization factor is used or if an additional internalization factor is used, i.e. if the combination complex was applied, it was determined, especially by the target cells, for example by specific surface antigens or receptors, specific to the type of cell that thus enables the targeted transfer of nucleic acid to that type of cell.

Nucleic acid affinity substances that can be used according to the invention include, for example, homologous organic polycations, such as polylysine, polyarginine, polyornithine, or heterogeneous polycations having two or more different positively charged amino acids, and these polycations may have chains of different lengths, and also non-peptide synthetic polycations such as polyethyleneimine. Other substances with nucleic acid affinity that are suitable are naturally occurring DNA-binding proteins of a polycationic nature, such as histrons or protamines or analogs or fragments thereof, as well as spermine or spermidines.

The length of the polycation is not critical, as long as the complexes are essentially electroneutral. A polylysine chain length range of about 20 to about 1000 lysine monomers is advantageous. However, for a given length of DNA, there is no critical polycation length. When DNA consists of 6,000 bp and 12,000 negative charges, the amount of polycations per mole of DNA can be,

60 moles of polylysine 200

30 moles of polylysine 400; or

120 moles of polylysine 100, etc.

Those who are normally trained in quite routine experimentation can choose other combinations of polycation length and molar amount.

Other suitable nucleic acid affinity substances as part of the conjugate are intercalating substances such as ethidium dimers, acridine or intercalating peptides containing tryptophan and/or tyrosine and/or phenylalanine.

Regarding the qualitative composition of the nucleic acid complex, the nucleic acid to be transferred into the cell is generally determined first. Nucleic acid is defined primarily by the biological impact to be achieved in the cell and, in the case of use for gene therapy, by the gene or part of the gene that should be expressed, for example for the purpose of replacing a damaged gene or by a targeted sequence of genes that should be inhibited. Nucleic acids that will be transported into the cell can be DNA or RNA, while there are no restrictions on the nucleotide sequence. If the invention is applied to tumor cells, for the purpose of their use as a cancer vaccine, the DNA to be introduced into the cell is usually designated as an immuno-modulating substance, eg a cytokine such as IL-2, IL-4, IFN gamma, TNF alpha. DNA combinations that insert a cytokine can be particularly useful, eg IL-2 and IFN gamma. Another useful gene for introducing into tumor cells can be the "multi drug resistance gene" mdr. It is also possible to introduce into the cell two or more different nucleic acid sequences, eg a cDNA containing plasmid, which encodes two different proteins under the control of suitable regulatory sequences, or two different plasmid constructs containing different cDNAs.

Therapeutically effective inhibitory nucleic acids for delivery into cells to inhibit specific gene sequences include gene constructs from which antisense-RNA or ribozymes have been translated. In addition, it is also possible to introduce oligonucleotides into cells. Anti sense nucleotides usually comprise 15 or more nucleotides. Optionally, oligonucleotides can be multimerized. When iribozymes are to be introduced into a cell, they are usually introduced as part of a gene construct, which contains stabilizing gene elements, eg tRNA gene elements. The gene constructions of this team were published in EP A 0 387 775.

Regardless of the nucleic acid molecules that inhibit genes, eg viral genes, genes with different mode of inhibitory action can be used, given their complementarity. Examples are genes designated as viral proteins, which have so-called trans-dominant mutations (Herskowitz, 1987). Gene expression in the cell creates proteins that dominate the corresponding progenitor protein and thus protect the cells, which acquire "cellular immunity" by inhibiting viral renewal.

Trans-dominant mutations of viral proteins required for replication and expression are suitable, eg, Gag-, Tat-, and Rev-mutants, which have been shown to inhibit HIV replication (Trono et al., 1989; Green et al., 1989; Malim et al., 1989).

Another mechanism for achieving intracellular immunity involves the expression of RNA molecules that contain a site for connecting an essential viral protein, for example the so-called TAR baits (Sullenger et al., 1990).

Examples of genes that can be used in somatic gene therapy and that can be transferred into cells as components of a gene construct, using the present invention, include factor VIII (hemophilia A) (see, e.g., Wood et al., 1984), factor IX (hemophilia B) (see, e.g. Kurachi, K. et al., 1982), adenosine deaminase (SCID) (see, e.g. Vlerio, D. et al., 1984), -1 antitrypsin (pulmonary emphysema) (see, e.g. . Ciliberto, G. et al., 1985) or the cystic fibrosis transmembrane conductance regulatory gene (see, for example, Riordan, J.R. et al., 1989). These examples are not limiting in any way.

As for the size of nucleic acids, a large range is possible. Gene constructs from about 0.15 kb (in the case of a ribozyme-containing tRNA gene) to about 50 kb or more can be transferred into cells by the present invention. Smaller nucleic acid molecules can be used as oligonucleotides.

It is clear that the widest possibility of application is made possible precisely by the fact that the present invention is not subject to any limitation to the number of genes and by the fact that very large gene constructs can also be transferred via the invention.

Starting from nucleic acid, a substance that has an affinity for nucleic acid, especially an organic polycationic substance, is determined to ensure the complexation of nucleic acid, with the fact that the resulting complexes are essentially electroneutral. If the complexes contain a conjugate of an additional internalizing factor and a substance having an affinity for nucleic acid, the polycationic component of the conjugate is taken into account in relation to the electroneutrality spectrum. During the earlier inventions, it was found that optimal transfer of nucleic acid into the cell can be achieved if the ratio of the conjugate to the nucleic acid is chosen so that the internalizing factor-polycation/nucleic acid complexes are substantially electroneutral. It was found that the amount of nucleic acid accepted into the cell does not decrease, if some of the transferrin-polycation conjugate is replaced by a non-covalently linked polycation. In some cases there may even be a substantial increase in DNA uptake (Wagner et al., 1991a). It was observed that the DNA of the complex is present in a form pressed into toroidal structures with a diameter of 80 to 100 nm. The amount of polycations is therefore chosen, considering the two electroneutrality parameters of achieving a compact structure, while the amount of polycations resulting from the charge of nucleic xylin, considering the achieved electroneutrality, generally also guarantees the compactness of DNA.

Thus, in a further embodiment of the invention, the complexes are also substances that bind nucleic acid in a non-covalently bound form, which may be the same or different from the binding factor. In the event that the endosomolytic agent is a free vvirus, the complexes contain nucleic xylin and an internalizing factor conjugate. In the event that an endosomolytic, eg viral conjugate is used, the nucleic acid is complexed with this conjugate, in accordance with the conjugate of the additional internalizing factor. The choice of non-covalently bound "free" substitutions, which have an affinity for nucleic acid, according to their nature and quantity, is also determined by the conjugate(s), especially taking into account the linking factor contained in the conjugate; if, for example, the linking factor is a substance that has no or limited capacity for DNA condensation, it is generally advisable, with regard to achieving efficient internalization of the complex, to use substances that have an affinity for DNA, and that have this property to a large extent. If the binding factor alone of the substance that condenses the nucleic acid and has already achieved compaction of the nucleic acid is sufficient for efficient internalization, it is advisable to use a substance that has an affinity for the nucleic acid, which leads to increased expression based on other mechanisms.

Suitable "free" substances that have an affinity for nucleic acid xylin, according to the invention, include compounds capable of condensing (condensing) nucleic acid and/or protecting it from undesirable degradation in cells, especially substances of a polycationic nature that were mentioned before. The second group of suitable substances includes those which, by binding to the nucleic acid, contribute to the improvement of its translation/expression, by improving the accessibility of the nucleic acid to the expression mechanism of the cell. An example of a substance of this type is the chromosomal non-histone protein HMG1, which has been found to have the capacity to make DNA compact and contribute to cell expression.

With regard to complexes, when determining the molar ratios of an endosomolytic agent and/or internalizing factor to a substance that has an affinity for nucleic acid (or acids), it should be borne in mind that nucleic acid (acid) complexation occurs, that the created complexes can bind to the cell and internalize and that, either by itself or with the help of an endosomolytic agent, it is released from the endosomes.

Relationship internalizing factor: connecting factor; nucleic acid depends particularly on the size of the polycationic molecules and on the number and distribution of positively charged groups, whose criteria correspond to the size and structure of the nucleic acid (acids) to be transported. It is advantageous for the molar ratio of the internalizing factor to the substance having an affinity for the nucleic acid to be in the range of about 10/1 to about 1/10.

After constructing and synthesizing the conjugate and determining the optimal conjugate:DNA ratio for effective transfection, the amount of conjugate that can be replaced, if desired, by a free substance having an affinity for the nucleic acid can be determined by titration. If polycations are used both as binding factors and as a similar substance having an affinity for nucleic xylene, the polycations may be identical or different.

For the realization of the invention that uses viral conjugates, a method suitable for determining the ratio of the components contained in the complexes can consist primarily in determining the construction of the gene to be introduced into the cells, and then, as described above, in finding a virus or a viral component suitable for a particular transfection. Then the virus or viral component binds to the polycation and complexes with the gene construct. Starting from a certain amount of viral conjugate, titrations can be performed by treating target cells with that (constant) amount of conjugate and reducing DNA concentrations, or vice versa. In this way, the optimal ratio of DNA: virus conjugate was determined. If an additional internalizing factor is used, the procedure can be performed, for example, to determine the optimal ratio of viral conjugate to internalizing factor conjugate starting from a constant amount of DNA, by titration.

Complexes can be prepared by mixing the components of 1) nucleic acid, 2) viral conjugate of choice, 3) internalizing factor/binding factor conjugate, and optionally, 4) non-covalently bound substance that has an affinity for nucleic acid, all of which can be present in the form of diluted solution. If polycations are used as a binding factor and at the same time as "free" polycations, it is usually recommended first of all to prepare a conjugate mixture with "free" polycations and then combine this mixture with DNA. The optimal ratio of DNA: conjugate (i): polycation is determined by means of titration, i.e. by using a constant amount of DNA in a series of transfection experiments, and by increasing the amount of the conjugate/polycation mixture. The optimal ratio of conjugates to cations in the mixture is achieved by routine experiments or by comparing the optimal proportions of mixtures used in titration experiments.

DNA complexes can be prepared in saline concentrations. Another possibility is to use high salt concentrations (about 2 M NaCl) and then adjust to physiological conditions by slow dilution or dialysis.

The most suitable sequence for mixing nucleic acid components, conjugates, possibly non-covalently bound substances with affinity for nucleic acid, was determined by previous experiments. In some cases it may prove advisable to first complex the nucleic xylin with the conjugate(s) and then add a "free" substance that has an affinity for the nucleic acid, for example, a polycation, eg in the case of transferrin-ethidium dimer and polylysine conjugates.

In a favorable embodiment of the invention, the internalizing factor, that is, the additional internalizing factor is transferrin, and the binding factor is a polycation. The term "transferrin" refers to natural transferons as well as those modifications of transferrin that are bound by a receptor and transported into the cell.

Nucleic acid is accepted in the form of complexes in which internalizing factor-polycation conjugates are complexed with nucleic acid. If the composition includes a non-covalently bound substance that has an affinity for nucleic acid, it is usually 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", nucleic acid is internalized in the form of complexes in which internalizing factor conjugates, on the one hand, and endosomolytic conjugates, on the other hand, are complexed with nucleic xylin.

Conjugates of internalizing factor and polycation, which are used together with free virus or together with viral conjugates in combination complexes, can be prepared by a chemical method or a recombinant method, if the polycation is a polypeptide. The methods were first performed by the invention of EP 388 758.

Typically, within the scope of the present invention, conjugates have been used in which a glycoprotein, eg, transferrin, and a binding factor are linked to each other via one or more carbohydrate chains of the glycoprotein.

In contrast to conjugates prepared by conventional coupling methods, conjugates of this type are free from modifications induced by the binding substances used. In the case of glycoproteins that have only one or only a few carbohydrate groups suitable for coupling, eg transferrin, these conjugates also have the advantage that they are precisely determined with respect to their binding position to glycoproteins/coupling factor.

A suitable method for the preparation of glycoprotein-polycation conjugates is published in German patent application P 41 15 038.4; recently described by Wagner et al., 1991b.

The amount of endosomolytic agent used and its concentration depend on the individual transfections undertaken. It is preferable to use the minimum amount of virus or viral conjugate necessary to ensure the internalization of the virus (conjugate) and nucleic acid complex and its release from endosomes. The amount of virus is adjusted to the individual type of cells, and above all, the infectivity of the virus for that type of cells should be taken into account. Another criterion is a particular conjugate of internalizing factor and binding factor, especially with regard to internalizing factor, for which the target cell has a specific number of receptors. In addition, the amount of virus (conjugate) will depend on the amount of DNA to be introduced. In general, a small amount of virus is sufficient for stable transfection that requires only a small amount of DNA, while transient transfection, which requires large amounts of DNA, requires a large amount of virus. For a particular application, tests are performed with target cells intended for transfection, preferably with a mixed population of cells, and a vector system intended for transfection, in order to determine the optimal concentration of the virus by titration, while the DNA construction of the gene is used, which largely agrees with that intended for the specific use, given the size, and contains a reference gene for easier measurement of gene transfer efficiency. Within the scope of the present invention, geneluciferase and β-galactosidase have proven to be suitable as reference genes for such tests.

Another aspect of the invention relates to the process of introducing a nucleic acid complex, a nucleic acid binding substance and, if desired, an internalizing factor into multiple eukaryotic cells. The method is characterized by bringing the cells into contact with an agent that has the ability to be internalized into the cells, either by itself or as a component of the nucleic acid xylin complex, and to release the contents of the endosomes, in which the nucleic acid complexes are located, into the cytoplasm.

In general, it is best that the nucleic xylin complexes and the endosomolytic agent are administered together, but they can also be administered one after the other. In the case of separate applications, the order of application is not critical as long as the stages are carried out in short succession to ensure that the components will be in effective mutual contact.

In the case of using a free virus in a separate procedure, the simultaneous addition of a free virus preparation with complexes can be ensured, if the viral preparation is a transfection medium containing a nucleic acid complex.

In the case of simultaneous addition of free virus, the nucleic acid complexes and the virus preparation are mixed together before they are added.

In another embodiment, the endosomolytic agent is a component of the combination complex. To enhance gene expression, the compositions of the present invention can be added in multiple repetitions.

In another embodiment, the cells are primarily tumor cells. In a particularly favorable embodiment, the nucleic acid is xylene DNA that contains one or more sequences labeled as an immunomodulating substance, usually a cytokine.

In another embodiment, the cells are myoblasts, usually primary myoblasts.

In another embodiment, the cells are fibroblasts, usually primary fibroblasts.

In another embodiment, the cells are hepatocrates, usually primary hepatocytes.

In another embodiment, the cells are primary endothelial cells.

In another embodiment, the cells are primary epithelial cells.

Table 1 shows transfections of the present invention exemplified with a variety of different cell types.

The composition of the invention was also tested for the transfection of canine hemophilia B fibroblasts. Luciferase and β-galactosidase can be successfully expressed in these cells. In addition, the system was used to deliver 1.4 kb canine factor IX cDNA into fibroblasts from canine hemophilia. Canine factor IX can be detected in ELISA sandwich 24 hours after transfection.

In some cases, it is advisable to use a lysosomotropic substance with an endosomolytic agent, for example, if the agent is an endosomolytic peptide conjugate or a retrovirus, whose endosomolytic activities are not strictly dependent on acidic pH.

It is known that lysosomatotropic substances inhibit the activity of proteases and nucleases and therefore can inhibit the degradation of nucleic acids (Luthmann and Mangusson, 1983). These substances include chloroquine, monensin, nigericin and methylamine.

Monensin has been shown to contribute to an increase in the expression of the reference gene when Moloney retrovirus is used.

The presence of chloroquine can lead to the expression of the reference gene, introduced by transferrin-driven DNA admixture, in almost 100% of K562 cells. BNL.CL2 or HepG2 hepatocytes did not respond to chloroquine as well as K562 cells, but they could be transfected to a level of 5-10% when the endosomotyl properties of added, damaged or chemically inactivated free adenovirus were exploited.

By means of the present invention, the advantages of biological vectors are increased. As a result of receptor distribution, there is tropism for both the internalizing factor and the virus. By matching these two components with a particular population of cells, it is possible to achieve greater selectivity, which is of particular importance in the therapeutic application of this invention.

On the other hand, the present invention relates to pharmaceutical compositions containing as an active component a therapeutically active nucleic acid complex, usually as part of a gene construct, an endosomolytic agent (optionally conjugated) and, possibly, an internalizing factor conjugate. Any pharmaceutically acceptable carrier, such as saline or phosphate buffered saline, or any such carrier in which the DNA complexes are well soluble, may be used for use in the method of the present invention. References are published in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA, Osol (ed.) (1980), relating to methods of formulating pharmaceutical compositions.

The present invention offers the advantage of the greatest possible flexibility in application, among others as a pharmaceutical composition. The composition of the invention can appear as a lyophilizate or in a suitable buffer in a deep-frozen state. It may also be provided as a ready-to-use reagent in solution, usually cooled in solution. Optionally, components required for transfection, i.e. DNA, endosomolytic agent, conjugates or ready for conjugation with separate conjugation partner, DNA binding substance, optionally conjugated with internalizing factor, optionally free polycation, may be present in a separate buffer solution, or partially separated as an integral part of the transfection kit, which is also the subject of the present invention. Different DNA acids can be present in such a transfection kit, for example representing different antigens, and/or different internalizing factor conjugates, so that the transfection kit could be used as an adaptable system. Whether the constituents are present as a "ready-to-use" preparation or separately, to be mixed immediately before use, depends, regardless of the specific application, on the stability of the complex, which can be determined by routine stability tests. Adenovirus-polylysine conjugate linked by transglutaminase, which has proven to be stable during storage, is used as an integral part of the kit. In another advantageous embodiment, steptavidin-polylysine biotinylated adenoviruses are mixed before use. A number of different transfection kits can be devised by the ordinarily skilled practitioner to take advantage of the flexibility of the invention.

For therapeutic purposes, the composition can be administered systemically, usually intravenously, as part of a pharmaceutical composition. The target organs for this application can be, for example, the liver, spleen, lungs, bone marrow and tumors.

An example of local application is lung tissue (use of the composition according to the invention as part of a pharmaceutical composition in liquid form for infusion or as an aerosol for inhalation). In addition, the pharmaceutical compositions of the invention can be administered by direct injection into the liver, muscle tissue, into a tumor or by local application into the intestinal tract. A further method of administration of the pharmaceutical composition is administration via the biliary drainage system. This method of application enables direct access to the membranes of hepatocytes in the bile ducts, preventing interaction of the composition with blood components.

Recently, the possibility of using myoblasts (immature muscle cells) for gene transfer into the muscle fibers of mice was demonstrated. Since myoblasts have been shown to secrete the gene product into the blood, this method may have a much wider application than treating genetic damage to muscle cells, such as the damage present in muscular dystrophy. Thus, derived myoblasts can be used to release gene products that act in the blood or are transported through the blood. Experiments in the present invention have shown that myoblast cultures as well as myotubes can be transfected particularly efficiently. The most successful medium for transfection contains combination complexes of biotinylated adenovirus, transferrin-polylysine and streptavidin-polylysine. In addition to the reference genes luciferase and β-galactosidase, factor VIII is expressed in muscle cells. In addition, chicken adenovirus CELO was used in combination complexes, which contain wheat germ agglutinin as an additional internalizing factor.

Therapeutic application can also be "ex vivo", whereby the treated cells, eg bone marrow, hepatocytes or myoblasts, are returned to the body (eg Ponder et al., 1991, Dhawan et al., 1991). Further ex vivo application of the present invention refers to the so-called "vaccine cancer". The basis of this therapeutic approach is the isolation of tumor cells from the patient, and the transfection of cells with cytokine-labeled DNA. The next phase may involve inactivating the cells, eg by irradiation, in such a way that they can no longer regenerate, but still express the cytokine. The genetically modified cells are then added to the patient from whom they were isolated, as a vaccine. In the vicinity of the vaccination site, secreted cytokines activate the immune system, among other things, by activating cytotoxic T cells. These activated cells are able to express their influence on other parts of the body and to attack untreated tumor cells. Thus, the risk of tumor recurrence and the development of metastasis is reduced. A suitable way of applying cancer vaccines for gene therapy was described by Rosenberg et al., 1992. Instead of the retroviral vectors proposed by Rosenberg, the gene transfer system according to the present invention can be used. In experiments of the present invention, primary melanoma cells were successfully transfected with a reference gene contained in combination complexes of polylyzed adenoviruses and transfein polylysine.

The present invention can also be used in experiments to determine the host immune response to a given antigen. Such experiments are based on gene transfer to antigen-expressed cells.

Antigen-specific cytotoxic T lymphocytes (CTL), which kill infected cells, play an important role in self-defense against viral infections or tumors. The interaction between a T-cell and an antigen-presenting cell (APC) is limited by human lymphocyte antigens (HLA). To study CTL killing of antigen-expressing cells in an in vitro CTL killing experiment, the antigen must be presented to the CTL in the correct HLA context, which usually means the target cell.

The CTL-killing experiment can be performed as follows:

APC cells are transformed by DNA assembly containing an antigen-tagged sequence. Antigen epitopes will be bound to MHC class I molecules and added to the cell surface as a target for a specific CTL response. Thus, after incubation with the patient's serum sample, depending on the presence of specific CTL lymphocytes, ACP cells will be destroyed. The schedule is measured by monitoring the release, for example, of radioactive chromium that was incorporated into the APV cells before the addition of serum.

Established protocols (Walkner et al., 1989) use B-LCLs introduced to express antigenic genes by transfection with recombinant caccinia viruses. However, the cells that express the antigen effectively die in about a day due to the destructive action of the vaccinia virus.

These difficulties can be overcome by CTL knockdown experiments using gene transfer systems, inventions for introducing antigen-tagged DNA structures, eg structures that tag HIV or tumor antigens, into fibroblasts to restore antigenic expression.

Primary fibroblasts are easily obtained from biopsies, are easily developed, and it has been shown that they can be transfected with a particularly high efficiency (about 50 to 70%) according to the present invention.

Such experiments are useful for the identification of epitopes observed on killer cells, with regard to the composition of vaccines. Furthermore, they can be usefully used to determine an individual's HLA-restricted immune response against a given antigen.

Since a high level of expression of the transferred genes can be achieved in almost all cells, the invention can be used for the production of recombinant proteins. However, there are no or few restrictions regarding the sequence or molecular weight of the transferred DNA. There is also a wide spectrum of cell types that can be transfected with the DNA constructs of the present invention. Therefore, almost any type of cell can be used for the production of recombinant proteins, which ensures that the recombinant protein is produced in a reliable and completely modified form that guarantees a high biological activity of the product.

Gene transfer into cells can be performed as demonstrated for luciferase and interferon alpha, and almost any gene construct that increases the desired protein production can be transferred. The desired protein product can be obtained from the transfected cell culture (or cell supernatant or appropriate cell homogenate, according to the protocol for the individual protein product), 24 hours to a week, or more, after transfection.

The use of gene transfer systems, according to the current ism for the production of recombinant proteins, has the following advantages:

1) Due to the high efficiency of transfection (more than 90% of transfected cells can express the transferred gene to a large extent), it is not necessary to select positively transfected cells in advance and there is no need to establish stable cell lines. Small-scale cell culture may be sufficient to produce usable amounts of protein.

2) Large gene constructs can be transferred, and up to 48 kb have been successfully transferred so far.

3) Gene expression can be expressed in cells that promise appropriate processing and modification (eg, vitamin K-dependent carboxylation of clotting factors, see: Armentano, et al., 1990, or cell type-specific glycosylation).

4) The use of this method enables a wider selection of target cell types for gene expression.

Having generally described the invention, it will be further understood by reference to the following examples, which are set forth by way of illustration, but are not intended to be in any way limiting. All patents and publications listed here are presented in their entirety.

EXAMPLES

In the following Examples illustrating the present invention, the following materials and methods were used (unless otherwise indicated).

Preparation of the transferrin-polylysine/DNA complex

a) Human transferrin-polylysine conjugates

The method described by Wagner et al., 1991b, was used, according to which polylysine was attached to the chains of the carbohydrate side of transferrin. A solution of 280 mg (3.5 µmol) of human transferrin (iron-free, Sigma) in 6 ml of 30 mM Na-acetate buffer, pH 5, was cooled to 0ºC and 750 µl of 30 mM Na-acetate buffer, pH 5, containing 11 mg (51 µmol) sodium periodate. The mixture is left for 90 minutes in the dark on an ice bath.

In order to remove low-molecular products, gel filtration (Sephadex G-25, Pharmacia) was performed, which gives a solution containing about 250 mg of oxidized transferrin (measured by the ninhydrin test). (To show the oxidized form, which contains aldehydes and gives a colored reaction with anisaldehyde, samples were added dropwise to a silica gel thin-layer plate and dried, and the plates were then immersed in p-anisaldehyde/sulfuric acid/ethanol (1 /1/18), dried and heated). A solution of modified transferrin was added rapidly (within 10 to 15 minutes) to a solution containing 1.5 µmol of fluorescein-labeled poly(l)lysine with an average chain length of 190 lysine monomers, in 4.5 ml of 10 mM sodium acetate pH 5. The pH of the solution was adjust to pH 7.5 by adding 1 M sodium bicarbonate buffer. 4 portions of 28.5 mg (450µmol) of sodium cyanoborohydride are added to the mixture at intervals of one hour. After 17 hours, 2 ml of 5M sodium chloride was added to bring the total concentration of the solution to 0.75M. The reaction mixture is added to a column with a cation exchanger (Pharmacia Mono S HR 10/10) and eluted with a salt gradient from 0.75M to 2.5M sodium chloride with a constant content of 25 mM HEPES, pH 7.3. A high salt concentration when added to the column and at the beginning of the gradient is essential for obtaining polycationic conjugates. Some transferrin (about 30%) is eluted with the flow along with weak fluorescent activity. The bulk of the fluoroscein-labeled conjugate eluted at salt concentrations between 1.35M and 1.9M, and was captured in three fractions. these fractions (in the order in which they were eluted) gave after two dialysis according to 2 lit. 25M HEPES.a pH 7.3, fraction A (TfpL190A) containing 45 mg (0.56 µmol) transferrin, modified with 366 µmol polylysine, fraction B (TfpL190B) containing 72 mg (0.90 µmol) transferrin , modified with 557 µmol of polylysine and fraction C (TfpL190C), which contained 7 mg (85 nmole) of transferrin, modified with 225 nmole of polylysine. If not used immediately, conjugates are frozen with liquid nitrogen and stored at -20ºC in “iron-free” form. Before iron administration, samples (0.5 to 1 mg) were adjusted to physiological salt concentration (150 mM) with sodium chloride. Iron is introduced by adding 4 µl of 10 mM iron(III) citrate buffer (containing 200 mM citrate, adjusted to pH 7.8 with the addition of sodium bicarbonate) per milligram of transferrin content. Iron-containing conjugates were divided into small aliquots before use for DNA complexation, then frozen in liquid nitrogen or a mixture of dry ice and ethanol, and stored at -20ºC. (This procedure proved convenient, but repeated thawing and freezing was found to cause loss of conjugate activity.

b) Rodent transferrin-polylysine conjugates

A similar method was used as for human transferrin, in that the coupling was performed via the lanane of the carbohydrate side. Conjugates of 15.5 nmol rodent transferrin and 13 umol pL290 were obtained from 4.1 mg (51 nmol) rodent transferrin and 2.1 mg (34 nmol) pL290.

Plasmid-DNA

a) pRSVL-DNA

The DNA plasmid pRSVL (containing the Photinus pyralis luciferase gene under the control of the Rous Sarcoma Virus LTR Enhancer/Promoter (Uchida et al., 1977, De Wet et al., 1987)) was prepared using the Triton-x Lysis standard method (Maniatis), and then Cs/EtBr equilibrium centrifugation by density gradient decolorization with butanol and dialysis against 10 mM Tris/HCl pH 7.5 1 mM EDTA). For complex formation, generally, 6 µg DNA plasmid material in 350 µl HBS (150 mM NaCl, 20 mM HEPES, pH 7.3) is mixed with 12 µg transferrin-polylysine conjugate in 150 µl HBS, 30 min before addition to the cells.

b) pCMVL-DNA

Plasmid pCMVL (reference gene construct containing the Photinus pyralis luciferase gene under the control of the cytomegalovirus promoter) was prepared by removing the BamHI-Insert of plasmid pSTCX556 (Severne et al., 1988), the plasmid was treated with the Klenow fragment and HindIII/SspI and the Klenow-treated fragment from the plasmid pRSVL, containing the sequence indicated for luciferase, was introduced. pCMV gal was described by MacGregor and Caskey (1989). The DNA preparation was made analogously to pRSVL.

Production of viral preparations

a) Adenovirus preparations

The adenovirus strain d1312, described by Jones and Shenk, 1979, which is deficient in the Ela region, was used. Virus recovery was performed in the Ela- trans -complementing cell line 293, and large-scale purification was performed as described by Davidson and Hassell, 1987. Purified virus was stored in buffer (100 mM Tris, pH 8.0, 100 mM NaCl , 0.1%, BSA, 50% glycerol) or in HBS/40% glycerol, and aliquots were stored at -70ºC. The virion concentration was determined by UV-spectrophotometric analysis of the extracted genomic viral DNA

(Formula: one optical density unit, OD, A260, corresponds to 1012 viral particles/ml) (Chardonnet and Dales, 1970).

(b) Preparation of retroviruses

Moloney murine leukemia retrovirus N2 was packaged using the ecotropic packaging method (Keller et al., 1985, Armentano et al., 1987). Supernatants of virally expressed cells were collected, frozen in liquid nitrogen and stored at -20ºC. The supernatants used in the Examples had a titer of approximately 10 6 cfu/ml, as measured by neomycin-resistant colony formation with NIH3T3 cells. For virus concentration experiments, supernatants were passed through a 300 kD impermeable membrane (FILTRON) in an AMICON cell concentrator, under nitrogen pressure. This method normally concentrates 10 to 30 ml of supernatant ten times.

Stations and feedlots

HeLa cells were cultured in DMEM medium supplemented with 5% heat-inactivated fetal calf serum (FCS), 100 IU/ml penicillin, 100 µg/ml streptomycin and 2 mM glutamine. WI-38, MRC-5 and KB cells were cultured in EMEM medium (Eagle's Modified Essential Medium), supplemented with 10% heat-inactivated FCS, antibiotics, such as DMEM medium, 10 mM non-essential amino acids and 2 mM glutamine . CFT1, a respiratory cystic fibrosis epithelial cell line (prepared by the method described by Yankaskas et al., 1991; the CFT1 cell line is characterized by being homozygous for the CF-mutation F508 decay) was cultured in F12-7x medium (Willumsen et al. , 1989). For gene transfer experiments, cells were cultured in 6 cm plates until they were 50% confluent (5 x 10 5 cells). The medium was removed and 1 ml of DMEM or EMEM/2% FCS medium was added. Conjugate-DNA complexes were then added, followed immediately by adenovirus d1312 (0.05 - 3.2 x 104 particles per cell) or a comparable volume of virus storage buffer (1 - 80 µl). The plates were returned for one hour to the incubator (5% CO2, 37ºC) and then 3 ml of complete medium was added. After a further 24 hours of incubation, the cells were collected to measure the expression of the luciferase gene. In the case of CFT1, cells were cultured for 4 hours in F12-7X medium without human transfer, before the gene transfer experiment.

The following cell lines were obtained from ATCC and can be obtained under the catalog numbers given. HeLa cells: CCL 2, K562 cells: CCL 243, HepG2 cells: HB 8065, TIB-73-cells: TIB 73 (BNL CL.2), NIH3T3 cells: CRL 1658, 293 cells: CRL 1573, KB cells: CCL 17 , Wi-38 stations: CCL 75, MRC 5 stations CCL171. H9 cells were obtained from the AIDS Research and Reference Reagent Program, U.S. Department of Health and Human Services, Catalog number 87.

Primary lymphocytes were obtained by taking 25 ml of umbilical cord blood into test tubes containing EDTA. Aliquots were poured with 4.5 ml of Ficoll-Hypaque reagent (Pharmacia) and centrifuged for 15 minutes at 2500 rpm. The brown layer between the upper plasma layer and the clear Ficoll layer is removed (about 10 ml). 40 ml of IMDM with 10% FCS was added, the sample was centrifuged at 1200 rpm for 15 minutes, and the cell pellet was suspended in 50 ml of fresh IMDM with 10% FCS (the pellet density was about 2 x 106 cells/ml). 250 ul of phytohemagglutinin (PHA P, DIFCO) is added, the culture is incubated for 48 hours at 37ºC and with 5% CO2, recombinant IL-2 (BMB) is added at a concentration of 20 units/ml. Cells are then split 1:3 with IMDM/20% FCS, 2 units/ml IL-2. Aliquots of cells were frozen in liquid nitrogen in FCS with 5% DMSO. Before use, cells were propagated in IMDM with 20% FCS and 2 units/ml of IL-2.

For further binding studies HeLa cells were maintained at 4ºC in 1 ml DMEM supplemented with 2% FCS. Conjugate-DNA complexes were added, as in other assays, and plates were incubated for 2 h at 4ºC. The plates are then thoroughly washed with ice-cold DMEM/2% FCS, and then 2 ml of this medium is added. Adenovirus d1312 or virus buffer is then added and the cells are allowed to warm slowly before being placed in the incubator for a further 24 hours. After this incubation, the cells are collected and tested for the expression of the luciferase gene.

The luciferase experiment

Preparation of cell extracts, standardization of the necessary content and determination of luciferase activity are performed as described by Zenke et al., 1990, Cotten et al., 1990, and EP 388 758.

Example 1 - Determination of the effect of adenovirus treatment on gene transfer by transferrin-polylysine conjugates.

First of all, the effect of increasing the virus dose on the ability of a certain amount of the conjugate-DNA complex to achieve gene transfer was examined. To create the complex, mix 6 µg of plasmid pRSVL with 12 µg of human transferrin-polylysine conjugate (hTfpL190B). The conjugate-DNA complex with different amounts of adenovirus d1312 (0.05 - 3.2 x 104 virus particles per cell) was added to HeLa cells. The results of this analysis are shown in Figure 1. Luciferase activity is expressed in units of 50 µg of total cellular protein. According to this analysis, an increase in the amount of added adenovirus leads to an increase in gene transfer. Figure shows average values from 2 to 4 separate experiments. The spaced lines show the standard deviations.

Example 2 - Dosing effect of the conjugate-DNA complex

Logarithmic dilutions of the conjugate-DNA complex, prepared as in Example 1, were added to HeLa cells, with or without the addition of a constant dose of adenovirus d1312 (1 x 104 viral particles per cell). Luciferase activity was determined as in Example 1. The results are shown in Figure 2.

Example 3 - Enhancement of gene transfer caused by transferrin polylysine by adenovirus, occurs via receptor-directed endocytosis.

a) The effect of adenoviral treatment on the transfer of compacted DNA.

The following components were used for transfection:

6 µg of pRSVL-DNA without transferrin-polylysine conjugate (DNA); 6 µg of pRSVL-DNA plus 6 µg of unconjugated polylysine 270 (DNA+pL); 6 µg of PRSVL-DNA with 12 µg of the transferrin-polylysine conjugate used in the previous examples (DNA + HTfpL190B). these transfection materials were added to HeLa cells with or without adenovirus d1312 (1 x 104 viral particles per cell. Preparation of cell extracts, standardization of total protein, and determination of luficerase activity were performed as in previous examples. The results of the tests are shown in Figure 3A.

b) The effect of adenovirus treatment on the transfer of receptor-bound DNA.

Conjugate-DNA complexes (DNA + hTfpL190B) or polylysine-DNA complexes (DNA + pL) were associated with HeLa, without internalization, by incubation at 4ºC. Unbound complex was removed prior to the addition of adenovirus d1312 (d1312) (1 x 104 viral particles per cell) or an appropriate volume of buffer. After that, incubation was performed at 37ºC, to enable the internalization of bound DNA complexes and adenoviruses. Luciferase activity was determined as described (Figure 38).

c) Effect of adenovirus treatment on gene transfer by transferrin-polylysine conjugates.

Conjugate-DNA complexes containing 6 µg of pRSVL-DNA and in addition 12 µg of transferrin-polylysine (DNA +hTfpL190B) were added to HeLa cells with 104 adenovirus particles (d1312) per cell or the corresponding amount of heat-inactivated adenovirus d1312 (d1312 h.i.). Thermal inactivation was performed by incubating for 30 minutes at 45ºC (Defer et al., 1990). Figure 3C shows luciferase activity.

Example 4 - Effect of adenovirus treatment on gene transfer by transferrin-polylysine conjugates in selected cell lines.

Conjugate-DNA complexes (6 µg pRSVL + 12 µg hTfpL190B) were added to cells of the cell lines CFT1, KB, HeLa, WI38 and MRC5, with or without adenovirus d1312 (1 x 104 virus particles per cell). The efficiency of gene transfer for different cell lines was determined as in the previous examples by the luciferase experiment (Figure 4).

Example 5 - Enhancement of luciferase gene expression functions at the level of gene transfer and not at the level of transactivation

The cell line designated K562 10/6, which constitutively expresses luciferase, was prepared by transfecting the cells with a plasmid containing a fragment of the RSV luciferase gene (Apal/Pvul fragment pRSVL; De Wet et al., 1987), cloned into the ClaI position of the pUCµ Locus ( Collis et al., 1990). this plasmid was complexed with a transferrin-polylysine conjugate, and K562 cells were transfected with these complexes, according to the method described by Cotten et al., 1990. Since the pUCµ Locus plasmid contains a neomycin resistance gene, it is possible to select for clones expressing luciferase , based on neomycin resistance. K562 10/6 clone was selected for further experiments.

Aliquots of the parental cell line K562 (in 200 µl RPMI 1640 with 2% FCS; 500,000 cells per sample) were treated with either 12 µg TfpL plus 6 µg pRSVL, or 4 µg pL 90 plus 6 µg pRSVL, in 500 µl HBS in both cases. Specified amounts of adenovirus d1312 (Figure 5) were allowed to act on the cells for 1.5 hours at 37ºC, after which 2 ml RIMI and 10% FCS were added. Then the incubation is continued at 37ºC for another 24 hours and then the cells are prepared

to measure luciferase activity. Incubation with adenovirus was found to lead to a significant increase in luciferase activity (Figure 5A). This applies to both TfpI complexes (2000 light units compared to 25,000 light units) and pL 90 complexes (0 compared to 1.9 x 106 light units). This shows that the K562 cell line has the ability to internalize the pRSVL polylysine complex and that this internalization, as measured by luciferase expression, is significantly increased by the presence of adenovirus.

Analogous tests were performed with K561 10/6 cells, which naturally express the RSVI luciferase gene, and similar amounts of adenovirus d1312 were used. Aliquots of 500,000 cells (in 200 µl RPMI with 2% FCS) were inhibited at 37ºC for 1.5 h, with the amounts of adenovirus d1312 indicated in FIG. 5B. Then, as in the parental cell line, RPMI plus 10% FCS is added, inhibition is continued for 24 hours, and luciferase activity is determined. As seen in Figure 5B, treatment of these cells with adenovirus had no detectable effect on luciferase activity. The control values are in the same range as the values for the virus-treated samples.

Example 6 - Transfection of liver cells with asialofetuin-polylysine (AFpL) conjugates or with tetra-galactose peptide-pL (gal 4pL) conjugates in the presence of adenovirus.

a) Preparation of lactosylated peptide

3.5 mg (1.92 umole) of the branched peptide Lys-(N-Liz)Lys-Gly-Ser- Gly-Gly-Ser - Gly-Gly-Ser- Gly-Gly-Cys, prepared by the Fmoc method using "Applied Biosystems 431A Peptide Synthesizer, containing a dithiopyridine group for Cys, was treated with a solution of 7.85 mg of lactose in 40 µl of 10 mM aqueous sodium acetate pH 5, at 37ºC. 4 aliquots of 0.6 mg (10 µmol) of sodium cyanoborohydride are added to the solution, at intervals of about 10 hours. After a total of 64 hours at 37ºC, 0.5 ml of HEPES pH 7.3 and 15 mg of dithiothreitol (DTT) are added. Fractionation by gel filtration (Sephadex G-10, 12 y 130 mm. Eluent: 20 mM NaCl) under argon gave 3.6 ml of lactosylated peptide solution in free mercapto form (1.74 µmol corresponding to Ellmann test yield 84%). Samples of the modified peptide showed a staining reaction with anisaldehyde, but no reaction with ninhydrin. This agrees with the assumption that all four terminal amino groups are lactosylated. The tetra-galactose peptide-polylysine conjugate is shown in Figure 6.

b) Preparation of polyzine modified with 3-dithioiridine propionate.

400 µl of a 15 mM ethanolic solution of SPDP (6.0 µmol) was added, with vigorous stirring, to a gel-filtered solution of 0.60 µmol of poly-L-lysine with an average chain length of 290 lysine monomers (pL290, hydrobromide, Sigma) in 1, 2 ml 100 nm HEPES Ph 7.9. One hour later, 500 µl of 1M sodium acetate, pH 5, was added after gel-filtration (Sephadex G-15) with 100 mM sodium acetate, the solution contained 0.56 µmol of pL290 with 5.77 µmol of dithiopyridine bound.

c) Connection of the peptide with polylysine.

Conjugates were prepared by mixing 1.5 µmol of the lactosylated peptide prepared in a) in 3 ml of 20 mM NaCl with 0.146 µl of modified pL290 obtained from b) in 620 µl of 100 mM sodium-acetate buffer, under argon. After the addition of 100 µl of 2M HEPES, pH 7.9, the reaction mixture was left to stand for 18 hours at room temperature, with the addition of BNaCl the salt concentration was adjusted to 0.66 M and the conjugates were isolated by chromatography with a cation exchanger (Pharnacia Mono S column HR 5/ 5; elution gradient: Buffer A 50 mM HEPES pH 7.3; Buffer B: buffer A with 3 M NaCl). The product fractions were eluted with a salt concentration of about 1.2 M - 1.8 M, and were captured in two conjugate fractions: the conjugate fractions were named ga14pL1 and ga14pL2. Dialysis with 25 mM HEPES pH 7.3 gave a conjugate fraction of ga14pL1, with 24 nmoles of modified pL290 and ga13pL2 with 24.5 nmoles of modified pL290.

d) Preparation of asialofetuin conjugate

The conjugates were prepared on the same basis as the transferrin conjugates. A similar method for the preparation of asialoorosomucoid-polylysine conjugate was described by Wu and Wu in 1988.

Coupling of asialofetuin with polylysine was performed with the bifunctional reagent SPDP (Pharmacia). A solution of 100 mg (2.2 µmol) of asialofetuin (Sigma) in 2 ml of 100 mM HEPES pH 7.9 was subjected to gel filtration on a Sephadex G-25 column. 330 µm of a 15mM ethanol solution of SPDP (5.0 µmole) was added to 4 ml of the resulting solution, with vigorous mixing. After standing for one hour at room temperature, purification is performed by another gel filtration (Sephadex G-25); so that 5 ml of a solution of 1.4 µmol of asialofetuin, modified with 2.5 µmol of dithiopyridine binder, is 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; the same procedure was used to prepare the transferrin conjugate) in 6.5 ml 100mM HEPES pH 7.6, under nitrogen atmosphere. The reaction mixture is left for 24 hours at room temperature. The conjugates are isolated from the reaction mixture by chromatography on a cation exchanger (pharmacia Mon s-column HR 10/10; elution gradient, buffer A: 50 mM HEPES pH 7.9; buffer B: buffer A plus 3M sodium chloride) and sodium chloride is added until to reach a final concentration of 0.6 M, before addition to the column. The product fraction was eluted with a salt concentration of about 1.5 M. Dialysis with HBS gave conjugates, which contained 0.52 µmol of asialofetuin modified with 0.24 µmol of pL190.

e) Transfection of HepG2 cells with pRSVL-DNA complexes.

HepG2 cells were grown in DMEM medium plus 10% FCS, 100 IU/ml penicillin, 100 µg/ml steptomycin and 2 mM glutamine, in T25 vials. Transfections were performed at a density of 400,000 cells per vial. Before transfection, cells were washed with 4 ml of fresh medium containing 10% FCS. Immediately before transfection, chloroquine (Sigma) is added in such a way that the final concentration in the cell suspension (with DNA solution) is 100 µM.

10 µg of pRSVL-DNA in 330 µl HBS was mixed with the amounts of TfpL190B conjugate, asialofetuin pL90 conjugate (AFpL), polylysine 290 (pL) or tetra-galactosepeptide polylysine conjugate ga14pL, specified in Figure 7, in 170 µl HBS. In comparative experiments, it is added after 30 min. 40 µg of asialofetuin (gal)4pL +Af) or 30 µg of lactosylated peptide ((gal)4pL + (gal)4). The mixture is added to the cells. Cells are incubated at 37ºC for 4 hours, and then the transfection medium is replaced with 4 ml of fresh DMEM medium plus 10% FCS. After 24 hours the cells are collected for the luciferase experiment. The values given in Figure 7 show the total luciferase activity of the transfected cells. As the figure shows, pL and TfpL show weak luciferase activity. (gal)4pL shows values as high as AfpL. (gal)4 or Af compete for the asialoglycoprotein receptor and, as expected, decrease values.

f) Transfection of HepG2 cells with pCMVL-DNA complexes.

HepG2 cells were grown on 6 cm plates, to a density of 300,000 cells per plate, as described under e). Before transfection, the cells were washed with 1 ml of fresh medium containing 2% FCS.

6 µg of pCMVL-DNA in HBS was mixed with amounts of TfpL10B conjugate (TfpL), asialofetuin-pL conjugate (AfpL), polylysine 290 (pLys290), (gal)4pL1 or (gal)4pL2, as specified in Figure 8, in 170 µl HBS. After 30 minutes, 1 ml of DMEM containing 2% FCS and 50 µl of standard adenovirus d1312C solution were added to each DNA-conjugate complex. In parallel experiments, 30 µg of the lactosylated peptide (gal)4pL ((gal)4pL1 + (gal)4 or (gal)4pL2 + (gal)4) was added, as specified. The mixture is added to the cells. The cells are incubated for 2 h at 37ºC, and then 1.5 ml of medium containing 10% FCS is added. Two hours later, the transfection medium was replaced with 4 ml of fresh DMEM medium plus 10% FCS. After 24 hours the cells are collected for the luciferase experiment. The values in Figure 8 show the total luciferin activity of the transformed cells. pLys290 shows an effect, (gal)4pL shows a stronger effect; addition of (gal)4, which competes for the asialoglycoprotein receptor, reduces the values to those obtained for polylysine.

g) Transfection of TIB73 cells with pCMVL-DNA complexes.

Cells of the ATCC TIB73 rodent embryo liver cell line (BNL CL.2; Patek et al., 1978) were grown at 37ºC in a 5% Co2 atmosphere, in “high glucose” DMEM (0.4% glucose), supplemented with 10% of heat-inactivated FCS, containing 100 IU/ml penicillin, 100 µg/ml streptomycin 2 mM glutamine, in 6 cm plates.

Transfections were performed to a cell density of 300,000 cells per plate. Before transfection, cells were washed with 1 ml of fresh medium plus 2% FCS.

6 µg pCMVL in 300 µl HBS was mixed with specified amounts of rodent transferrin-polylysine 290 conjugate (mTfpL), asialofetuin-pL conjugate (AFpL), polylysine 290 (pLys290), (gal)4pL1 or (gal)4pL2, in 170 µl HBS. . After 30 minutes, 1 ml of DMEM, containing 2% FCS, and 50 µl of standard adenovirus d1312 solution were added to each DNA conjugate complex. The mixture is added to the cells, the cells are incubated for 2 hours at 37ºC, and then 1.5 ml of medium containing 10% FCS is added. Two hours later, the transfection medium was replaced with 4 ml of fresh medium. After 24 hours the cells are collected for the luciferase experiment. The values shown in Figure 9B show the total luciferase activity of the transfected cells.

For comparison, transfection was performed without adenovirus in the presence of chlorquine. Transfection was performed at a cell density of 300,000 cells per plate. Before transfection, the cells were washed with 1 ml of fresh medium containing 2% FCS. Immediately before transfection, chloroquine (Sigma) is added so much that the final concentration in the cell suspension (plus DNA solution) is 100 µM. 6 µg pCMVL-DNA in 330 µl HBS is mixed with specified amounts of mTfpL, AFpL, pLys290, (gal)4pL1 or (gal)4pL2, in 170 µl HBS. After 30 minutes, DNA complexes are injected into the cells. The cells are incubated for 2 hours at 37ºC, and then 1.5 ml of medium containing 10% FCS and 100 µM chloroquine are added. Two hours later, the transfection medium is replaced with 4 ml of fresh medium. After 24 hours the cells are harvested for luciferase measurement. The values obtained for luciferase activity are shown in Figure 9A.

Example 7 - Introduction of DNA into T cells.

a) Preparation of antiCD7 polylysine190 conjugate.

A solution of 1.3 mg of antiCD7 antibody (Immunotech) in 50 mM HEPES pH 7.9 was mixed with 49 µl of a 1 mM ethanol solution of SPDP (Pharmacia). One hour after standing at room temperature, the mixture is filtered through a Sephadex G-25 gel column (eluent 50 mM HEPES buffer pH 7.9), whereby 1.19 mg (7.5 nmol) of anti-CD7, modified with 33 nmol pyridyldithiopropionate group. Poly(L)lysine190, labeled with fluorescein FITC, was modified analogously to SPDP and was brought into a form modified with free mercapto groups, by treatment with dithiothreitol, and then by gel filtration. A solution of 1 nmol of polysine190 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 pH 7.9), with exclusion of oxygen, and left overnight at room temperature. The reaction mixture is adjusted to a content of about 0.6 M by adding a 5 M NacL solution. Isolation of the conjugate was performed by ion exchange chromatography (Mono S, Pharmacia, 50 mM HEPES pH 7.3 salt gradient 0.6 M to 3 M NaCl). After dialysis against HEPES (10 mM, pH 7.3), the corresponding conjugates were obtained, which consisted of 0.51 mg (3.2 nmol) of antiCD7-antibody, modified with 6.2 nmol of polylysine 190.

b) Preparation of gp120-polylysine190 conjugate.

The coupling was carried out by methods known from the literature, by thioether coupling after modification with N-hydroxysuccinimide ester of 6-maleimidocaproic acid (EMCS, Sigma) (Fujiwara et al., 1981). Tiether bound gp120-polylysine190 conjugates:

A solution of 2 mg of recombinant gp120 in 0.45 ml of 100 mM HEPES pH 7.9 was mixed with 17 µl of a 10 mM solution of EMCS in dimethylformamide. After one hour at room temperature, filtration is carried out through a Sephadex G-25 gel column (eluent 100 mM HEPES-buffer 7.9). The resulting solution (1.2 ml) reacts immediately, with the exclusion of oxygen, with a solution of 9.3 nmol of polylysine 190, labeled with fluorescein and modified with 30 nmol of mercapto groups (in 90 µl of 30 mM sodium acetate pH 5.0), and leave overnight at room temperature. The reaction mixture is adjusted to a content of about 0.6 M by the addition of 5 M NaCl. Conjugates are isolated by ion exchange chromatography (Mono S, Pharmacia 50 mM HEPES pH 7.3, salt gradient 0.6 M to 3 M NaCl). After fractionation and dialysis against 25 mM HEPES pH 7.3, 3 conjugate fractions A, B and C were obtained, which consisted of: 0.40 mg rgp120 modified with 1.9 nmol polylysine 190 (in the case of fraction A), or 0.25 mg rpg120 modified with 2.5 nmol polylysine 190 (fraction B), or 0.1 mg rgp120 modified with 1.6 nmol polylysine 190 (fraction C).

pCMVL-DNA (6 µg/sample) were complexed with specified amounts of polylysine90 or specified polylysine conjugates in 500 µl HBS. At the same time, aliquots of H9 cells (106 cells in 5 ml RPMI with 2% FCS) or primary human lymphocytes (3 x 106 cells in Dulbecco's medium, modified according to Iscove (IMDM) with 2% FCS) were prepared. Polylysine-DNA complexes were added to each cell sample. After 5 minutes, the amount of adenovirus d1312 was determined. The cells were then incubated for 1.5 h at 37ºC, and then 15 ml of RPMI (in the case of H9 cells) or IMDM (in the case of primary lymphocytes) plus 20% FCS was added to each sample. Cells are incubated for 24 hours at 37ºC, harvested for processing as in other examples for determination of luciferase activity. The results of the performed tests are given in Fig. 10A (H9 cells) and Fig. 10B (primary lymphocytes): in H9 cells, the antiCD7 conjugate (Fig. 10A, columns 7 to 9) and the gp120 conjugate (Fig. 10A, columns 10 to 12) showed the best results, regarding gene transfer, achieved with adenovirus, while the gp120 conjugate achieved a clearer expression of the luciferase gene even in the absence of adenovirus. It is worth noting that in the tests performed, only the gp120 conjugate had the ability to introduce DNA into primary lymphocytes, and only in the presence of damaged adenovirus (Figure 10B, columns 7 and 8).

Example 8 - Adenovirus inactivation

a) UV inactivation

The d1312 adenovirus preparation, prepared and stored as described in the introductory section of the Primer, is placed in a well, 2 cm in diameter, of a cell culture plate (300 µl per well) on ice, 8 cm away from two UV lamps (Philips TUV15, G15 T8). The virus was exposed to UV radiation for the times specified in Figure 11A, and aliquots of each preparation were assayed for their viral titer, to determine whether, and to what extent, they were able to enhance gene transfer with polylysine-transferrin conjugates in HeLa cells.

Cultivation of cells and transfection was carried out mainly as written above from "cells and feeders". Components used for transfection are shown in Figure 11A. Complexes of pCMVL and 12 µg of TfpL were prepared in 500 µl of HBS and added to 3x105 HeLa cells (in 1 ml of DMEM plus 2% FCS. About 5 min later, 54 µl of each viral preparation was added to each culture and the culture was incubated at 37ºC for 1, 5 to 2 hours. Then a 5 ml aliquot of DMEM plus 10% FCS is added to each culture, incubation is continued at 37ºC for 24 hours and the cultures are harvested and assayed for luciferase activity. A 54 µl amount of non-irradiated virus is not within saturation, i.e., the assay is sensitive to an amount of virus that is at least threefold higher. The results obtained for luciferase expression are shown in Figure 11B (dashed rectangles). The viral titer of each preparation was determined using the "Ela complement cell line 293". Serial dilutions of unlabeled and labeled virus samples were plated in DMEM plus 2% FCS. In parallel, samples of 5x104 293 cells (in 2 cm wells) were plated in 200 µl DMEM plus 2% FCS. A 5 µl aliquot of each dilution was placed in each well To enable s e virus binds to the cells, incubation at 37ºC for 1.5 h was performed, and then 2 ml of DMEM plus 10% FCS was added to each well. After 48 hours, the cultures are examined to determine the cytopathic effect. The above virus dilution, in which less than 50% of cells in culture show a significant cytopathic effect after 48 hours, indicates the relative amount of infectious virus in each virus preparation. The obtained results are shown in Figure 11B (empty rectangles). The results of the tests performed in this example show that a 4-log decrease in viral titer, as a result of UV irradiation, is associated with only a twenty-fold decrease in luciferase gene transfer. This shows that the mechanisms, which are crucial for viral infectivity, can be destroyed without affecting the ability of the virus to enhance gene transfer.

It was observed that at low doses of virus, the increase in gene transfer caused by the virus decreases somewhat (Figure 11A, columns 3 to 6) and that this effect is more significant at higher doses (columns 7 to 10).

b) Inactivation of adenovirus with formaldehyde.

2 ml of the adenovirus preparation is passed through a 10 ml G25 column (Pharmacia Sephadex G 25, PD10), prepared with 150 mM NaCl, 25 mM HEPES pH 7.9, 10% glycerol and made up to a volume of 2.5 ml. Aliquots of the gel-filtered virus preparation were incubated for 20 hours on ice, without formaldehyde (O), with 0.01%, 0.1% or 1% formaldehyde. Tris pH 7.4 is then added to give a concentration of 100 mM, and then the samples are dialyzed, first against 1 liter of 150 mM NaCl, 50 mM Tris pH 7.4, and 50% glycerol, and then, overnight, against 2 x 1 liter 150 mM NaCl, 20 mM HEPES pH 7.9 and 50% glycerol.

Aliquots of virus were then assayed for their titer on 293 cells (CPE end point assay, or “plaque” assay, Precious and Russell, 1985). Then, the influence of formaldehyde-treated viruses on gene transfer was determined in HeLa cells (300,000), as in previous examples, by measuring luciferase activity. 90 µl of the viral preparation resulted in DNA transfer, which corresponded to more than 108 light units. Treatment of the virus with 0.01% or 0.1% formaldehyde resulted in a weak reduction in gene transfer activity (approximately a fourfold reduction at 0.1%). Although treatment with 1% formaldehyde caused a striking loss of gene transfer activity, 90 µl of virus was still sufficient to produce gene expression corresponding to 104 light units.

When treated with 0.1% formaldehyde, the reduction of viral titer to 105 PFU (plaque-forming units) was associated with a reduction of luciferase activity by only 10%. The test results are shown in Figure 12A.

c) Adenovirus inactivation with long-wave UV radiation + 8-methoxy psoralen.

Aliquots of the purified virus were adjusted to 0.33 µg/µl with 8-methoxy psoralen (the standard concentration is 33 µg/µl of 8-methoxy psoralen dissolved in DSMO) and were exposed to a source of UV radiation with a wavelength of 365 nm (UVP model TL-33). , on ice, at a distance of 4 cm from the lamp filter. Exposure to UV light lasted 15–30 minutes, as indicated in the figure legend. Viral samples were then passed through a Sephadex G-25 column (Pharnacia, PD-10), prepared with HBS + 40% glycerol and stored at -70ºC.

Viral preparations were tested either for their activity in increasing the release of pCMVL/hTfpL conjugates in HeLa cells (as evident by the score of light units of luciferase activity, Fig. 12B, right ordinate) or for their ability to restore in 293 cells (viral titer, left ordinate of Fig. 12B).

In the examples that follow, which show an increase in the internalization of the transferrin-polylysine-DNA complex using retroviruses, the following methods and materials were used:

Transferrin-polylysine 190 conjugates and conjugate-DNA complexes were prepared in the same way as in the previous examples. NIH3T3 cells were grown on DMEM medium, supplemented with 10% FCS, 100 i.u. penicillin, 100 µg(ml streptomycin and 2 mM glutamine. For transfection, 5 to 7x105 cells per T25 were isolated from the plate 18 to 24 hours before transfection. Immediately before transfection, cells were placed on fresh medium and various components used for transfection were added, in the following order: chloroquine (100 uM, where indicated), polylysine-transferrin-DNA complex and retovirus preparation. The cells were then incubated for 4 hours at 37ºC, the medium was changed and the cells were harvested after 24 hours. The extracts were prepared in three freezing cycles - melting Aliquots of the extract, standardized for protein content, were tested for luciferase activity, as indicated in the previous examples.

Example 9 - Transfection of NIH 3T3 cells with Moloney virus.

Under the conditions indicated, transfections of 106 NIH3T3 cells were performed with TfpL-DNA complexes in the presence of 100 µM chloroquine, or without chloroquine, as shown in Figure 13. It was found that without chloroquine, the values for luciferase activity reached barely the baseline (column 1). , while in the presence of chloroquine a high expression of the pRSVL reference gene was measured (column 2). Increasing amounts of Maloney leukemia virus (labeled RVS in the image), which were added to the cells at the same time as the DNA complexes, were able to further increase the expression of the luciferase gene.

Example 10 - Research on whether the influence of gene transfer in the transfection of NIH3T3 cells with transferrin-polylysine DNA complexes can be attributed to Moloney virus.

The viral preparation used in Example 9 was a crude, unfractionated supernatant of retrovirus-expressing cells. In order to gain insight into whether the increase in DNA transfer achieved with this viral preparation could indeed be attributed to the virus, the supernatant was subjected to dialysis/concentration purification as described above, with the retroviral supernatant (shown as RVS in the drawing) being concentrated by a factor of 10. If a retrovirus is responsible for the increase, the activity retained by the membrane should, regardless of any inactivation of the extremely unstable retrovirus during the concentration phase, be approximately 10 times greater than the starting supernatant. As in the previous example, 106NIH3T3 cells were transfected under the conditions given in Figure 14. Figure 14 shows that the increasing effect on gene transfer is present in the membrane retentate (20 to 600 µl was used, columns 3-6). It was also found that 200 to 600 ul of a tenfold concentrate preparation is about half as active as 2 to 6 ml of the original, unconcentrated retrovirus preparation (columns 7 and 8). Parallel tests were performed with human K562 cells, which do not have receptors for ecotropic rodent retroviruses. As expected, there was no increase in gene expression.

Example 11 - Interactions between transferrin and its receptor play a role in the effect of Moloney virus on gene transfer.

In order to exclude the possibility that the transfer of the TfpL/pRSVL complex in the cell can be attributed to the non-specific binding of polylysine to the retrovirus, and with the aim of explaining the entry mechanism in more detail, the ability of the retrovirus to transport plasmid DNA, complexed only with polylysine, into the cell was examined. The amount of required polylysine corresponds to the optimum amount determined earlier, which contributes to the total condensation of plasmid DNA, and is similar to the amount of polylysine required for the polylysine-transferrin conjugate (Wagner et al., 1991). The tests, the results of which are shown in Figure 15, showed that the reference gene, in the absence of chloroquine, was not expressed either in the form of the TfpL-pRSVL complex, or in the form of the pL-pRSVL complex (columns 1 and 2). In the presence of retrovirus, on the other hand, reference DNA, administered as a TfpL complex, was expressed, but not as a pL-DNA complex (see columns 3 and 4 together with columns 5 and 6). In addition, assays performed showed that the presence of excess free transferrin resulted in a reduction in retrovirus-facilitated DNA transfer (columns 7 and 8). These results confirm the assumption that the interaction between transferrin and its receptor plays an essential role in increasing the uptake of DNA, which is affected by the retrovirus.

Example 12 - Influence of pH on the effect of retroviruses on gene transfer.

The experiments performed in this example were performed with the aim of examining the effect of pH on the ability of retroviruses to enhance gene transfer. Transfection experiments were performed as in previous examples. Two well-rated inhibitors of endosomal pH reduction, monensin and ammonium chloride, were used. The results of the experiment are shown in Figure 16. The influence of these two substances on TfpL-DNA transfer was tested and it was found that none of them could adequately replace chloroquine. However, higher concentrations of ammonium chloride were found to slightly increase the expression of the luciferase gene (columns 1 to 5). The retrovirus itself shows a slight increase in DNA transfer, as observed in previous examples (column 6). A sharp increase was observed when the retrovirus was used in the presence of 1 µM monensin (column 7). A smaller effect was observed at higher concentrations of monensin (column 8) and in the presence of ammonium chloride (columns 9 and 10).

Example 13 - increase in gene transfer achieved by transferrin conjugates using the N-terminal endosomolytic peptide of influenza HA2 hemagglutinin.

a) Peptide synthesis

Peptide sequence (SEQ ID NO:1) Gly-Leu-Phe-Glu-Ala-Ile-Ala-Gly-Phe-Ile-Glu-Asn-Gly-Trp-Glu-Gly-Met-Ile-Asp-Gly-Gly -Gly-Cys, was synthesized by the Fmoc (fluorenylmethoxycarbonyl) method (Atherton et al., 1979), using the Applied Biosystems 431A Peptide Synthesizer. Side chain protecting groups were t-butyl for Cys, Glu and Asp and trityl for Asn. After the coupling reaction, a ninhydrin test was performed, which showed a coupling level of more than 98% for each phase. Double couplings were performed starting at amino acid 19. The N-terminal Fmoc group was removed from a portion of the peptide resin with 20% piperidine in NMP (N-methylpiperidine). Then the Fmoc fractions, protected and unprotected, were washed with DCM (dichloromethane) and dried under high vacuum. The yields were 294 mg of F-moc-free peptide resin and 367 mg of F-moc-protected peptide resin. An amount of 111 mg of Fmoc-free peptide resin was subjected to trifluoroacetic acid cleavage for up to 1.5 h using a mixture of 10 ml TFA, 0.75 g phenol, 300 µl EDT (ethanedithiol), 250 µl Et-S-Me (ethylmethylsulfide) and 500 µl of water. The peptide is filtered from the resin through a glass sinter filter. The resin is washed with DCM and added to the filtrate. The filtrate is concentrated to about 2 ml and then added dropwise to 40 ml of ether with stirring. The peptide precipitate is removed by centrifugation, and the ether supernatant is discarded. The precipitate is washed three times with 40 ml of ether and dried under high vacuum. 58 mg of the obtained crude product is dissolved in 3.5 ml of 20 mM NH4HCO3, which contains 300 µl of 25% NH3/1. The solution is gel-filtered using the same buffer on a pre-packed Sephadex G-25 column (Pharmacia PD-10). All material was applied to a Mono Q column (Pharmacia); gradient: C10 min 100% A, 10-100 min 0-100% B. A: 20 mM NH4HCO3 + 300 ul NH3/1. B: A + 3 M NaCl. Measured at 280 nm, Trp-fluorescence detected at 354 nm. Flow rate: 1 ml/min. The product was eluted with 1 M NaCl. The main fraction of the Mono Q column was further purified by reverse phase HPLC (high pressure liquid chromatography) with a BIORAD-HI-Pore RP-304 column (250x10 mm); gradient 50 to 100% buffer B in 12.5 min, 12.5 to 25 min 100% B; A: 20mM NH4HCO3 + 300 µl NH3/1, B: A in 98% methanol. Flow rate: 3 ml/min. Measured at 237 nm. The product was eluted to 100% B. The product fractions were evaporated on a Speedovac evaporator, redissolved in buffer A and finally lyophilized. A yield of 8.4 mg of HPLC-purified product was obtained in the form protected by cysteine. This peptide is designated as P16. In order to obtain P16 in free mercapto form, the t-butyl protected substance is treated for 30 minutes at room temperature with thioanisole/ethanedithiol/trifluoroacetic acid/trifluoromethanesulfonic acid (2/1/40/3). Trifluoromethanesulfonic acid was added in the specified proportion, after the other components. The peptide was isolated by ether precipitation followed by gel filtration (Sephadex G-25) using the aforementioned buffer A, under an argon atmosphere.

b) Attachment of influenza peptide to polylysine

b1) Direct connection via SPDP (succinimidylpyridyldithiopropionate)

19.8 mg of polylysine 300 hydrobromide (Sigma) was gel-filtered on a Sephadex G-25 column (Pharmacia PD-10) in sodium acetate pH 5 to eliminate low molecular weight fractions. Based on the ninhydrin test, the pL concentration after gel filtration was 3.16 mg/ml. The pH value of the solution is adjusted to 7-8 with 1M maOH. 0.64 µmole of SPDP (Pharmacia: 40 mM solution in absolute ethanol) was added to 2.5 mlpL of solution (7.5 mg pL = 0.13 µmole). This corresponds to a SPDP to pL molar ratio of 5:1. The mixture is left to react overnight and is gel-filtered in 20 mM NH4HCO3 pH 8.2, on a G-25 column. After reduction of 1 aliquot of the filtrate with DTT (dithiothreitol), measurement of thiopyridone showed that the reaction was complete.

0.3 µmole pL modified with PDP (on a mol PDP basis) in 2.2 ml, is allowed to react with 0.35 µmole peptide in thiol form. The white precipitate that occurs when the peptide and pL are mixed is dissolved by adjusting the solution to 2 M guanidinium hydrochloride, allowing the reaction to proceed overnight. Photometric measurement of thiopyridone in the reaction mixture again confirms that the reaction is complete. The mixture is then dialyzed twice against 2 liters of 20 mM HEPES/0.5 M guanidinium hydrochloride. The resulting solution is added to a Mono S column (0.7 x 6 cm, Pharmacia) (gradient: 0 to 20 min 100% A, 20 to 140 min 0-100% B. A: 20 mM HEPES pH 7.3/0 .5 M guanidinium hydrochloride, B: 20 mM HEPES pH 7.3/3 M guanidinium hydrochloride, 0.3 ml/min.Detection at 280 nm, and fluorescence detection at 354 nm, excitation at 280 nm). The product fraction, which was eluted with 1.5 M guanidinium hydrochloride, was dialyzed against 2 liters of HBS. After that, the determination of the pL concentration by the minhydrin test showed a concentration of about 1.14 ml/ml. The amount of peptide in the conjugate solution was calculated from its absorbance at 280 nm; this gave a peptide to pL molar ratio of 4:1.

b2) Connecting via a polyethylene glycol bond.

14.6 mg pL 300 hydrobromide (Sigma) was gel-filtered as described in b1). According to the ninhydrin test, the pL concentration after gel filtration was 4.93 mg/ml. The pH value of the solution was adjusted to 7 to 8 with 1 M NaOH. 4.33 µmol PDP (Pharmacia; 30 mM solution in absolute ethanol) was added to 2.7 ml pL solution (13.3 mg pL = 0.22 µmole). This corresponds to a PDP:pL molar ratio of 20:1. After 1.5 h, the reaction mixture is filtered in a Sephadex G-25 column in a 0.1 M sodium acetate/3 M guanidinium hydrochloride solution. After reduction of an aliquot of the filtrate with DTT, thiopyridone was determined, which showed that the product fraction contained 3.62 µmol of PDP. PDP-modified pL was reduced by the addition of 79 mg DTT. After 2 hours of reduction, the solution is again filtered on G-25, under the specified conditions. The thiol test according to Ellaman showed a thiol concentration of 3.15 µmole in 2.224 ml.

In 500 µl of 20 mM NaHCO3/3 M guanidinium hydrochloride, 17.6 mg = 5 µmol of POE (polyoxyethylene-bis(6-aminohexyl), Sigma) is dissolved and treated with 13.8 mg of EMCS (N-hydroxysuccinimide ester -maleimidocaproic acid, Sigma) = 44.7 µmol, dissolved in dimethylformamide, 300 ul. After 30 minutes, the solution is gel-filtered on G-25 (20 mM NaHCO3/3 M guanidinium hydrochloride). Photometric measurement, at 300 nm, showed a concentration of 6.36 µmol of reacted EMCS in 2 ml of solution.

1.39 umole of peptide in thiol form (in 2.ml of 20 mM NaHCO3/3 M guanidinium hydrochloride) was added dropwise to 1.05 ml of this solution (corresponding to 3.34 umole of EMCS) with vigorous mixing of the mixture, in a stream of argon. After 15 minutes, the Ellman test could not detect the presence of free thiol groups. The solution of reduced, mercaptomodified pL is adjusted to pH 7-8 by adding 1 M NaOH. 1.37 ml of this solution was added to the above reaction mixture with vigorous stirring using a Vortex mixer. This gave a peptide-SH:POE-EMCS:pL-SH molar ratio of 1:2, 4:1.4 (based on EMCS and SH). After 2.5 h of reaction, the Ellman test could no longer prove the presence of thiol groups. The material was dialyzed overnight against 2 liters of 20 mM HEPES pH 7.3/0.6 M NaCl and then added to a Mono S column (gradient: O to 20 min 22%A, 20-150 min 22-100% B A: 20 mM HEPES pH 7.3, B: A + 3 M NaCl. Flow rate 0.3 ml/min. UV measurements were performed at 280 nm and fluorescence measurements at 354 nm). The product eluted with 1.5 - 1.6 M NaCl was dialyzed against 2 liters of HBS. Measurement of the pL concentration using the minhydrin test and photomaterial determination of the peptide concentration, at 280 nm, gave a calculated pL ratio of 12:1, with a pL concentration of 0.49 mg/ml in a total volume of 4.5 ml.

c) Preparation of liposomes

Liposomes were prepared by the REV (reverse phase evaporation) method (Szoka and Papahađopoulos, 1978; Straubinger and Papahađopoulos, 1983): aqueous phase 10 mM HEPES pH 7.3; 100 mM calcein/150 mM NaCl; organic phase: a solution of 300 µmol of L-α-lecithin (from egg yolk, mainly palmitoyloleoylphosphatidylcholine; Avanti Polar Lipids) in 260 µl of chloroform is evaporated on a rotary evaporator. The material is then dried under high vacuum and redissolved in 3 ml of diethyl ether. 1 ml of the aqueous phase is washed well with the ether phase using a Vortex mixer, and then it is treated with ultrasound for 5 min at 0ºC (in a bath-type "sonicator"). After 30 minutes on ice, the material is again treated with ultrasound for a further 10 minutes. The resulting stable emulsion is slowly evaporated on a rotary evaporator. After the diethyl ether has been removed at 100 mbar, 0.75 ml of the aqueous phase is added. The remaining traces of diethyl ether are removed by further evaporation at 50 mbar for 30 minutes. The resulting preparation was centrifuged at 500 rpm. Of this, 1 ml was squeezed through a "nucleopore" polycarbonate membrane (0.1 μm), resulting in a final volume of 0.7 ml of liposome solution. Liposomes were separated from extraneous material by gel filtration (Sephadex G-50, Pharmacia; 23 ml gel volume, 10 mM HEPES pH 7.3/150 mM NaCl). 6 fractions of 500 ul were collected. Lipid phosphorus was determined by the method of Bartlet, 1959, with 2 mM.

d) Liposome leakage experiment

Release of liposome contents (leakage) was measured by the release of incorporated calcein and the resulting dilution, which stops fluorescence quenching (Bondeson et al., 1984). Calcein fluorescence was measured with a KONTRON SMF 25 spectral fluorometer (excitation at 490 nm, emission at 515 nm). For this purpose, aliquots of the liposome solution were diluted 100 times with 0.1 M sodium acetate/50 mM NaCl or 10 mM HEPES/150 mM NaCl buffer of the appropriate pH value (4.3, 4.5, 5.0, 6.0, 7.3), to achieve a value of up to 1 ml. 2.5 µl peptide (t-butyl protected form; 1 µg/µl solution in HBS) was added to the solutions, in the cuvettes, under a gentle stream of argon (Final concentration 400 nM peptide). Casein fluorescence was measured at different time intervals after peptide addition. Values for 100% permeation were determined by addition of 2 µl Triton X-100 (Fluka).

The same procedure was used to measure the fluorescence of calcein after the addition of the peptide-pL conjugate to the liposome solution. 2.5 µg of conjugate (1 µg/µl concentration based on pL alone) was added to 1 ml of liposome solution (final concentration of 20 nM modified peptide). Similarly, 2.5 µg of the peptide-polylysine conjugate was subjected to a leak-through experiment, after incubation with 4 µg of DNA (15 minutes).

The peptide was found to cause liposome release only in the acidic region (Figure 17). The peptide conjugate was active at a significantly lower pH value, while even at neutral pH a strong activity was found, which further increased with decreasing pH value. Complexation of the conjugate with DNA eliminated the activity at neutral pH, while there was significant activity at acidic pH values.

e) Transfection of K562 cells

K562 cells were cultured in suspension in RPMI 1640 medium (Gibco BRL plus 2 g sodium bicarbonate/l) plus 10% FCS, 100 i.u. per ml penicillin, 100 µg/ml streptomycin and 2 mM glutamine, up to a density of 500,000 cells/ml. 12 to 15 hours before transfection, cells were placed in fresh medium containing 50 µM desferrioxamine (this measure was taken to increase the number of transferrin receptors). On the day of transfection, cells were harvested, suspended in fresh medium containing 10% FCS plus 50 µM desferrioxamine (250,000 cells per ml) and 2 ml aliquots were plated in a 24-well plate.

6 µg of pCMVL-DNA in 160 µl of HBS is mixed with the amounts of tFpL conjugate specified in Figure 18 or with pL300 in 160 µl of HBS, then after 15 minutes the specified amounts of influenza peptide-pL-conjugate (P16pL) are added, and after a further 15 minutes, the mixture is added to the K562 cells. Cells are incubated for 24 hours at 37ºC and then harvested for the luciferase assay. Luciferase activity was determined as described in earlier examples. The values given in Figure 18 represent the total luciferase activity of the transfected cells.

f) Transfection of HeLa cells

HeLa cells were cultured in 6 cm dishes, as described under “cells and feeders”. Transfections were performed at a density of 300,000 cells per plate. Before transfection, the cells were incubated with 1 ml of fresh medium containing 2% FCS. 6 µg pCMVL-DNA in 160 ml HBS was mixed with the amounts of TfpL conjugate specified in Figure 19, or with pL300 or a mixture of both in 160 µl HBS. After 15 minutes, the indicated amounts of influenza peptide-pL-conjugate (p16pL) are added, and after a further 15 minutes, the mixture is added to the cells. The cells are incubated for 2 hours at 37ºC, then 2.5 ml of fresh medium with the addition of 10% FCS is added. Cells are incubated for 24 hours at 37ºC and then harvested for the luciferase assay. Luciferase activity was determined as described in the previous examples. The values given in the figure represent the total luciferase activity of transfected cells.

Example 14 - Enhancement of gene transfer achieved by transferrin conjugates, using the second N-terminal endosomolytic peptide of influenza HA2 hemagglutinin

a) Synthesis of influenza peptide - polylysine conjugate

Peptide sequence (SEQ ID MO:2) Gly-Leu-Phe-Gly-Ala-Ile-Ala-Gyl-Phe-Ile-Gli-Asn-Gly-Trp-Glu-Gly-Met-Ile-Asp-Gly-Gly -Cys (marked P41), was synthesized in the same way as the peptide described in example 13,a). Connection of the influenza peptide with polylysine (pL300) was performed as in example 13, b1), by binding via SPDP. This resulted in conjugates (P41pL) with a molar ratio of peptide to pL of 4:1.

b) Transfection of HeLa cells.

HeLa cells were grown in DMEM medium 5% FCS, 100 i.u. of penicillin per ml, 100 µg/ml streptomycin and 2 mM glutamine, in 6 cm plates. Transfections were performed at a density of 300,000 cells per plate. Before transfection, the cells were incubated with 1.5 ml of fresh medium containing 2% FCS. 6 µg pCMVL-DNA in 160 µl HBS (150 mM NaCl, 20 mM HEPES 7.3) was mixed with 6 µg TfpL190B conjugate in 160 µl HBS, and after 15 min 10 µg peptide-pL-conjugate influenza P41pL or, for comparison, 18 µg of peptide-pL-conjugate influenza P16pL (see example 13), (Figure 20). The indicated amounts of the two peptide conjugates were verified as optimal amounts for increasing gene transfer. After a further 15 minutes, the mixture is added to the cells. The cells are incubated at 37ºC for 4 hours, then 2 ml of medium containing 18% FCS is added. After 24 hours the cells are harvested for the luciferase assay. The values shown in Figure 20 represent the total luciferase activity of the transfected cells.

Comparison of experiments with the two peptide conjugates shows a greater than 3.5-fold increase in gene transfer achieved with the second P41pL peptide conjugate.

c) Transfection of BNL CL.2 cells with influenza peptide-polylysine conjugates.

BNL CL.2 cells were cultured as described in example 6. Influenza P14 peptide was conjugated to polylysine 300 with a molar ratio of peptide to polylysine of 1:1, 3:1 and 8:1,

Complexes of 6 µg pCMVL DNA and 20 µg conjugate were added to the cells. For comparison, 20 µg of pL300 or 20 µg of P16 polylysine conjugate, prepared as described in Example 13, were used. Cells were incubated at 37ºC for 4 hours, and then 2 ml of medium containing 18% FCS was added. After 24 hours, the cells are harvested for the luciferase experiment, the results of which are shown in Figure 20B. The peptide content in the conjugates is related to the enhancement of gene expression. In liposome permeation experiments (Figure 20C), which were performed as described in experiment 13, the activity of the conjugates (at pH 5, corresponding to 2.5 µg of polylysine) increased with their peptide content. (in the picture P41 is marked with "influ2").

Example 15 - Transfection of HeLa cells with the β-galactosidase reference gene construct and "in situ" display of β-galactosidase expression.

a) Cultivation and transfection of cells.

For transfection, HeLa cells were cultured in DMEM medium containing 5% FCS, penicillin, streptomycin and glutamine, as described in the previous examples, in dishes with lids /vel. 3 cm). (3 x 104 cells per dish).

For transfection, 6 µg β-galactosidase reference gene construct (pCMV-β-gal) in 160 µl HBS is complexed with 12 µg TfpL190B in 160 µl HBS and incubated for 30 minutes at room temperature.

In another example, 6 µg pCMV-β-gal in 160 µl HBS is incubated with 6 µg TfpL190B in 80 µl HBS, for 15 minutes at room temperature. Then 12 µg of the influenza peptide conjugate (P16pL), prepared in Example 13 in 80 µl of HBS, was added and the mixture was incubated for a further 15 minutes. These DNA-polycation complexes are mixed with 1 ml of DMEM plus 2% FCS, antibiotics and glutamine, as described above. To demonstrate the effect of chloroquine and adenovirus on transfection success, in additional experiments, chloroquine was also added to the medium containing DNA polycation complexes, in a final concentration of 100 µmol or 50 µl of adenovirus strain d1312 solution.

For transfection, the original medium was removed from the cells and 1 ml of medium containing DNA complexes with or without chloroquine or virus was added. At the end of the incubation period of 2 hours at 37º, 1 ml of DMEM containing 10% FCS, antibiotics and glutamine was added to the cells and the incubation was continued for another two hours. Then the medium was removed and the cells were grown in 3 ml of fresh DMEM plus 10FCS, antibiotics and glutamine.

b) β-galactosidase experiment

48 hours after transfection the culture medium was removed, the cells were washed once with phosphate buffer solution (PBS) and fixed with 5% glutardialdehyde in PBS for 5 minutes at room temperature. Then the fixative is discarded, and the cells are washed once with PBS. Then, incubation with the staining solution (10 mM phosphate buffer pH 7.0, 150 mM NaCl, 1 mM MgCl2, 3.3 mM K4Fe(CN)6·3H2O, 3.3 mM K3Fe(CN)6 and 0.2% 5-bromo-4-chloro-3-indolyl-β-galactopyranoside), at 37ºC, 20 minutes to 3 hours (Lim and Chae, 1989). Then the coverslips are washed in PBS, water and 96% ethanol, dried and placed in "Mowiol" on glass slides. A Zeiss Axiophot microscope was used for analysis.

Figure 21 shows microscopic magnifications (112 times). A: HeLa cells transformed with 6 µg of pCMV-β-gal, complexed with 12 µg of TfpL190B. The staining reaction for β-galactosidase lasted for 3 hours. The image shows that only a few cells express the β-galactosidase gene (55 cells; colored cells are indicated by an arrow). B: HeLa cells transfected with 6 µg pCMV-β-gal, complexed with 6 µg TfpL190B and 12 µg P16pL. staining reaction: 3 hours. A few cells (250 cells) express β-galactosidase. However, the cell response is stronger than in A. C: HeLa cells transfected with 6 µg pCMV-β-gal, complexed with 6 µg TfpL190B and 12 µg Pl6pL in the presence of 100 µM chloroquine. Staining reaction. 3 hours. Numerous groups of cells show a very positive reaction (more than 1,000 cells). D: HeLa cells transfected with 6 µg of pCMV-β-gal, complexed with 12 µg of TfpL190 in the presence of adenovirus d1312. Staining reaction: 20 minutes. Almost all cells (more than 90%) show a positive reaction. E. Non-transfected HeLa cells (control for the specificity of the -galactosidase reaction).

Example 16 - Transfection of HeLa cells with a 48 kb cosmid in the presence of adenovirus.

a) Preparation of a cosmid containing a luciferase-tagged sequence.

A 3.0 kb Sal I fragment, containing a single P. pyralis luciferase-tagged sequence, under the control of the RSV promoter, was isolated from plasmid p220RSVLuca and ligated into the unique Sal I position of cosmid clone Cl-7aAl, to form concatamers. (Cl-7aAl captures a 37 kb human genomic DNA Sau3A fragment, which does not obviously tag genes, cloned into the BamHI site of the cosmic vector pWE15 (Stratagen). The product of the ligation reaction was then ligated “in vitro”, and the resulting phage particles were infected into E. coli NM544 and plated on LB amp plates. Recombinants were subjected to colony hybridization selection, using a 3.0 kb Sal I fragment (32p random smear labeled) as a hybridization probe, and numerous facts analyzed according to established restrictions. The structure of the cosmid (CosLuc), which contains a single sample of the Sal I insert, grown and purified on a CsCl gradient (total size = 48 kb).

The small control cosmid pWELuc (12 kb) was prepared by digesting CosLuc with Not I, religating, transforming the bacteria, and isolating a clone containing the appropriate plasmid. This resulted in a 12 kb DNA molecule lacking the human DNA insert and part of the CosLuc “polylinkee”. Plasmid pSPNeoLuc (8 kb) is the plasmid described in example 5, which contains a fragment of the RSV-luciferase gene (ApaI/Pvul fragment of pRSVL, cloned in the Cla 1 position of the pUCu Locus).

b) Cosmid release into HeLa cells.

HeLa cells (3 x 104 cells per 6 cm dish) were covered with 1 ml of DMEM + 2% FCS and incubated with TfpL/DNA complexes prepared as described in the chapter "Materials and methods", which contained the specified amounts of hTfpL, free pL and DNA. The cell incubation mixture contained, in addition, either 100 µg of chloroquine (columns 1 and 2) or 10 µl of adenovirus d1312 containing 5x1011 particles per ml, (columns 3-12). After 2 hours of incubation at 37º, add 4 ml of DMEM + 10% FCS to each container. After 24 hours the cells are collected (harvested) and the luciferase activity is measured. The results are shown in Figure 22A.

c) Cosmid release in Neuroblastoma cells.

Cells of the neuroblastoma cell line, labeled GIME-N (Donti et al. 1988) (1x106 cells per 6 cm dish), covered with 1 ml DMEM + 2% FCS, were incubated with TfpL/DNA complexes prepared as described in chapter "Materials and methods", which contained the specified amounts of hTfpL, free pL and DNA. Cell incubation mixtures contained, in addition, either 100 µM chloroquine (columns 3 and 4) or 10 µl adenovirus d1312, which contained 5 x 1011 particles per ml, (columns 5 and 6). After 2 hours of incubation at 37ºC, add 4 ml of DMEM + 10% FCS to each container. After 24 hours the cells are collected and the luciferase activity is measured. The results are shown in Figure 22B.

Example 17 - Gene transfer via chemically bound adenovirus-polylysine conjugates

a) Preparation of adenovirus-polylysine conjugate by chemical linking.

2.35 ml of a gel-filtered solution of adenovirus (Sephadex G-25 PD10.Pharmacia) (d1312, approx. 1011 particles) in 150 mM NaCl/25 mM HEPES, pH 7.9/10% glycerol, was mixed with 10 ul ( 10 nmol) of a 1 mM solution of SPDP (Pharmacia). After 3.5 hours at room temperature, the modified virus is separated from the excess reagent by gel filtration (as above). The solution is purged with argon and allowed to react, under exclusion of oxygen (under argon), with 42 µl of a solution of FITC-labeled polylysine (1 nmol), modified with 2.3 nmol of mercaptopropionate groups (prepared as described in EP 388 758). After standing for 18 hours at room temperature, half of the solution is transferred to a centrifuge tube, carefully covered with a solution of cesium chloride (density 1.33 g/ml) and centrifuged at room temperature for 2 hours at 35,000 rpm (SW60 rotor). The viral layer was collected as a 200 µl cesium chloride fraction and diluted to 1 ml with HBS/50% glycerol. The experiment shows that the DNA was derived from 200 µl of the modified virus;: the virus solution was diluted with 1 ml of HBS and mixed with 100 µl of the 35S-labeled DNA solution (15 ng of pRSVL, prepared by Mick transfer). As a control, the same experiment was performed in parallel with the same amount of unmodified d1312 virus. After 30 minutes, the samples were transferred to centrifuge tubes, carefully covered with 1 ml of cesium chloride solution (density 1.33 g/ml) and centrifuged for 2 hours at 35000 rpm (SW60 rotor) at room temperature. The gradient is divided into 5 fractions: fraction 1, 1 ml; fraction 2, 0.6 ml; fraction 3-5, each 200 µl. The radioactivity of 200 µl of all fractions was measured, and the results are shown in Figure 23. Fractions containing virus, especially fraction 3, show significantly higher radioactivity than the control fraction. This can be attributed to the specific binding of the polylysine-modified adenovirus to the labeled DNA, and the presence of cesium chloride may cause partial dissociation of the complex.

b) Transfection of K562 cells.

K-562 cells (ATCC CCL 243) were cultured in suspension in RPMI 1640 medium (Givco BRL, + 2g sodium bicarbonate/1 10% FCS, 100 U/ml penicillin, 100 µl/ul streptomycin and 2 mM glutamine) until density of 500,000 cells/ml. 12 to 20 hours before transfection, cells were placed in a medium containing 50 µM desferrioxamine (this measure was taken to increase the number of transferrin receptors). On the day of transfection, cells were harvested, suspended in fresh medium containing 10% FCS + 50 µM desferrioxamine (250,000 cells per ml) and 2 ml aliquots were plated in a 24-well plate.

Determined amounts of pCMVL-DVA (6, 0.06, 0.06 µg) in 100 µl HBS were mixed with 50 µl polylysine adenovirus (pLadeno) or with corresponding amounts (35 µl) of the control adenovirus (d1312). After 20 minutes, appropriate amounts (12, 1.2, 0.12 µg) of TfpL1=OB conjugate in 150 µl HBS are added. After a further 20 minutes, the mixture is added to the K562 cells. Cells are incubated for 24 hours at 37ºC and then harvested for the luciferase assay. Luciferase activity was determined as in the previous examples. The values given in Figure 24 represent the total luciferase activity of the transfected cells.

c) Transfection of HeLa cells.

One method of testing the activity of the polylysine-virus conjugate consists in checking the ability of the conjugate to transfer very small amounts of DNA (less than 0.1 µg). An increased DNA transfer capacity was expected, if the adenovirus is directly attached to the polylysine to condense the DNA, because the internalizing factors (transferrin and adenovirus (protein)) are directly attached to the DNA that will be transported. To test this assumption, a constant amount of polylysine-adenovirus conjugate (2.5 µl, about 5x107 viral particles) was complexed with varying amounts (3 to 0.0003 µg) of reference plasmid in 475 µl HBS. After 15 minutes of incubation at room temperature, an amount of transferrin-polylysine corresponding to the mass of DNA is added to each sample (this amount of TfpL was chosen, because it guarantees complete "wrapping" (electroneutrality) of 50% of the plasmid DNA, and at the same time provides space for binding of the virus-polylysine conjugate. After the addition of TfpL, the mixtures were incubated for 15 minutes, and then each mixture was placed in a 6 cm culture dish containing 300,000 HeLa cells in 1 ml of DMEM/2% FCS. The cells were then incubated for 1.5 h at 37ºC, and then 4 ml of DMEM/10% FCS is added. In parallel, equivalent amounts of DNA are complexed with twice the mass of TfpL (amount for complete DNA condensation) and used for gene transfer into HeLa cells (once alone and once with 25 ul of the adenovirus preparation d1312 which is not attached to polylysine).After 24 hours the cells are harvested, extracts are prepared and aliquots are tested for luciferase activity. The results of these tests are shown in Figure 25. In the absence of adenovirus, no to determine luciferase activity in an amount of DNA smaller than 0.3 µg. Adenovirus bound to polylysine, as well as unbound, performed well with large amounts of DNA (3 µg and 0.3 µg). However, with unbound adenovirus, an approximately one-hundred-fold drop in activity was shown with 0.03 µg, and only slight activity below that amount of DNA. In contrast, the polylysine-derived adenovirus retains its gene transfer capacity even with 0.0003 µg of DNA. This amount of DNA corresponds to the amount of 150 DNA molecules per cell and about one virus particle per cell.

Example 18 - Gene yield using adenoviruses enzymatically linked to polylysine.

a) Enzymatic reaction

2 ml of the adenovirus preparation (strain d1312; 5x1010 PFU/ml) was placed in a Sephadex G-25 gel filtration column (Pharmacia) buffered with 25 ml of reaction buffer (0.1 M Tris-HC1; pH 8.0 2 mM DTT, 30 % glycerol). Elution was performed with 3.5 ml of reaction buffer. The enzyme ligation reaction mixture consists of 1150 µl of the viral eluate fraction, 0.5 nmol of guinea pig liver transglutaminase (TG) (Sigma), 2 nmol or 20 nmol of polylysine 290, 10 n m CaCl2 and reaction buffer to a final volume of 1500 µl. The reaction is carried out for 1 hour at 37ºC, and then it is stopped by the addition of 30 µl of 0.5 M EDTA. In order to monitor the binding specificity, reaction mixtures were also prepared without transglutaminase. Unincorporated polylysine is separated from the virus by centrifugation in a CsCl gradient (density 1.33 g/ml; 170,000xg, 2 hours). The fraction containing the viruses is collected, mixed with an equal volume of glycerol, frozen in liquid nitrogen and stored at -70ºC.

b) Presentation of association of polylysine with adenoviruses.

The reaction is performed as described above, with polylysine labeled with J125 (with Bolton-Hunter reagent, Amersham). After centrifugation of the CsCl gradient, the viral fraction was removed and separated using another CsCl gradient. The gradient was then fractionated and the radioactivity of each fraction determined using a scintillation counter. As shown in Figure 26, it is clear that in the radioactive mixture with TG (d1312/TG-pL), the radioactive polylysine is accumulated in the viral fraction (virus). In the control mixture without TG (d1312/pL), there was no accumulation of radioactive polylysine in the viral fraction.

c) Testing polylysine-modified adenovirus fractions for their influence on transfection efficiency.

I) Stations and feeding grounds

For transfection, 5x105 cells (rodent hepatocytes; ATCC No.: TIB 73) were seeded in 6 cm dishes in DMEM with 10% heat-inactivated fetal calf serum (FCS), 2 mM glutamine, 100 i.u. per ml penicillin and 100 µg/ml streptomycin.

II) Creation of the virus-DNA-transferrin complex.

50 µl of the polylysine-modified virus fraction was mixed with 6 µg of pCMVL plasmid DNA in 10 µl of HBS and incubated for 20 minutes at room temperature. Then 8 µg of mouse transferrin-polylysine 290B (mTfpL) is added to the mixture and further incubated for another 20 minutes.

III) Transfection of mouse hepatocytes

Virus.DNA-transferrin complexes are mixed with 1.5 ml of substrate (DMEM with 2% FCS, 2 mM glutamine and antibiotics) and added to the cells, after removing the old substrate. After 2 hours of incubation at 37ºC, 2 ml of DMEM with 10% FCS, glutamine and antibiotics are added to the cells. After another two hours of cultivation, the entire substrate is removed and 4 ml of fresh DMEM with 10% FCS, glutamine and antibiotics are added to the cells.

IV) Determination of luciferase expression.

24 hours after transfection cells were harvested and the luciferase assay was performed as described above. As can be seen from Figure 17, the virus preparation in which adenoviruses were treated with TG and 20 nmoles of polylysine (d1312/TG-20 nmoles pL) showed the strongest expression of 153540000 “light units”). The viral preparation with TG and 2 nmoles of polylysine (d1312/TG-1 nmole pL) is slightly less active (57880000 light units). A control fraction in which adenoviruses were treated with 20 nmol polylysine but no TG was less effective, approximately by a factor of 500. For comparison, further complexes were used for transfection with an adenovirus primer treated with both TG and no polylysine (d1312 ). This preparation gave a yield of 440300 l.j.

d) Increasing the transfection efficiency of polylysine-modified adenoviruses compared to unmodified adenoviruses, especially with small amounts of DNA

Transfection was performed as described in example c), with 50 µl of adenovirus fraction d1312/TG-20 nmol pL and 6 µg pCMV-Luc/8 µg mTfpL, 0.06 µg pCMVL(=pCMV-Luc)/0.8 µg mTfpL or 0.06 µg pCMV-Luc/0.08 µg mTfpL for complexation. For comparison, transfections were also performed with 6 µg, 0.6 µg, 0.06 µg pCMV-Luc/mTfpL complex and unmodified adenoviruses (d1312). Complexes with polylysine-modified adenoviruses were found to give high levels of expression, even with small amounts of DNA, while with unmodified adenoviruses expression was greatly reduced (Figure 28).

Example 19 - Gene transfer with conjugates in which the connection between adenovirus and polylysine is achieved via a biotin-streptavidin bridge

a) Biotinylation of adenovirus d1312.

2.4 ml of a gel-filtered solution (Sephadex G-25 PD102, Pharmacia) of adenovirus d1312 (about 1011 particles) in 150 mM NaCl/5 mM HEPES, pH 7.9/10% glycerol, was mixed with 10 µl (10 nmol ) 1 mM solution of NHS-LC biotin (Pierce 21335). After standing for 3 hours at room temperature, the biotin-modified virus is separated from the excess reagent by gel filtration (as above). The solution is adjusted to a glycerol concentration of 40% by the addition of glycerol (total volume 3.2 ml) and stored at -25ºC. Biotinylation of the virus was proven by a qualitative experiment, after the addition, drop by drop, of different dilutions to the cellulose nitrate membrane. After drying at 80ºC, 2 hours in a vacuum dryer, after "blocking" with BSA, incubation with streptovidin conjugated alkaline phosphatase (BRL), washing and 1 hour incubation with NBT/X-phosphate developing solution (nitro-blue-tetrazolium sol/ 5 bromo-4-chloro-3-indolyl phosphate, toluidine salt, Boehringen Mannheim), a positive staining reaction was obtained.

b) Preparation of streptovidin-polylysine conjugate.

Coupling of streptovidin to polylysine was performed by the method described by Wagner et al., 1990, and in EP-Al 388 758. 79 nmoles (4.7 mg) of streptavidin in 1 ml of 20 mM HEPES pH 7.9 and 300 mM NaCl were treated with a 15 mM ethanolic solution of SPDP (236 nmoles). After 1.5 h at room temperature, the modified protein gel is filtered through a Sephadex G-15 column, whereby 75 nmoles of streptovidin modified with 196 nmoles of a dithiopyridine linker are obtained. The modified protein is reacted under an argon atmosphere with 3-mercaptopropionate modified polylysine (75 nmol, average chain length of 290 lysine monomers, modified with 190 nmol mercapto-propionate linker) in 2.6 ml of 100 mM HEPES pH 7.9, 150 mM NaCl . Conjugates were isolated by cation exchange chromatography on a Mono S HR5 column (Pharmacia). Gradient: 20-100% buffer B. Buffer A: 50 mM HEPES pH 7.9; buffer B: buffer A + 3 M sodium chloride). The product fraction was eluted with a salt concentration between 1.2 M and 1.7 M. Dialysis against HBS (20 mM HEPES pH 7.3, 150 mM NaCl) yielded a conjugate composed of 45 nmol streptavidin and 53 nmol polylysine.

c) Transfection of HeLa cells.

HeLa cells were grown on 6 cm dishes as described in Example 13. Transfections were performed at a density of 300,000 cells per plate. Before transfection, cells were incubated with 1 ml of fresh substrate containing 2% FCS.

6 µg pCMVL-DNA in 100 µl HBS, mixed with 0.8 µg streptovidin-polylysine in 170 µl HBS. After 20 minutes, 3 µg of polylysine pL300 in 170 µl of HBS is added. After another 20 minutes, 65 µl of biotidinylated adenovirus or, as a control, an appropriate amount of adenovirus d1312 (30 µl, initial virus for modification) is added. The complex mixture (“biotinAdV/complex A” or “control AdV”, see Figure 29) is allowed to stand for a further 20 minutes.

Another method of complexation was performed by mixing 65 µl of biotinylated adenovirus, first with 0.8 µg of streptovidin-polylysine in 50 µl of HBS, then by adding 6 µl of pCMVL-DNA in 170 µl of HBS, after 20 minutes, and after a further 20 minutes, by adding 3 µg polylysine pL300 in 200 µL HBS. (complex mixture (“biotinAdV/complex B).”

0.6 µg pCMVL-DNA in 67 µl HBS, mixed with 0.3 µg streptavidin-polylysine in 33 µl HBS. After 20 minutes, add 65 µl of biotinylated adenovirus or, as a control, an appropriate amount of adenovirus d1312 (30 µl, starting virus for modification). Complex mixtures (“biotinAdV/complex A” or “control AdV”, see Fig. 29) are allowed to stand for a further 20 min., and then diluted to 500 µl with HBS. Alternatively, complexation was performed by mixing 65 µl of biotinylated adenovirus, first with 0.3 µg of streptavidin-polylysine in 50 µl of HBS, after 20 minutes by adding 0.6 µg of pCMVL-DNA in 50 µl of HBS. The complex mixture (“biotinAdV/complex B)” is left for a further 20 minutes and then diluted to 500 µl with HBS.

The mixtures are added to the cells, the cells are incubated for 2 hours at 37ºC, and then 2.5 ml of fresh medium containing 10% GCS is added. The cells are incubated for 24 hours at 37ºC, and collected (harvested) for the luciferase experiment. Luciferase activity was determined as described in the previous examples. The values given in Figure 29 represent the total luciferase activity of the transfected cells.

In parallel, transfections of HeLa cells were carried out using a bitinylated virus conjugate as a viral component, which was inactivated by treatment with psoralen/UV-irradiation. Inactivation was performed as follows: a batch of 200 µl of biotinylated virus was placed in two wells of a 1.6 cm tissue culture plate. µl (33 mg/ml) 8-methoxypsoralen (in DMSO) is added to each sample, the container is placed on ice and irradiated for 10 minutes with a UV lamp (365 nm; UVP TL-33 lamp) at a distance of 4 cm from the sample to the filter . After irradiation, the two samples are combined and gel-filtered (G-50, Nick column, Pharmacia), through a column previously prepared with 40% glycerol in HBS. Aliquots of 75 µl were complexed with 0.8 µg streptavidin-polylysine and used to transfect HeLa cells as described above.

The cytopathic end point experiment found that the viral titer is reduced by a factor greater than 104 upon inactivation, while the transmission capacity is reduced by less than 50% at high concentrations and by a factor of 5 at low concentrations.

d) Fraction of K562 cells.

K562 cells are grown in suspension in RPMI 1640 substrate (Gibco BRL, +2g sodium bicarbonate per 1 liter) + 10% FCS, 100 units/ml penicillin, 100 µg/ml streptomycin and 1mM glutamine, and reach a density of 500,000 cells/ml. . At 16 hours before transfection, the cells were placed in a fresh substrate containing 50 µM desferrioxamine (Sigma). The morning after transfection, cells are harvested, resuspended in fresh medium containing 10% FCS (+50% µM desferrioxamine) to 250,000 cells per ml, and plated in a 24-well plate (2 ml per well).

Three different types of complexes were prepared: a) A solution of 6 µg pMCVL-DNA in 160 µl HBS (150 mM NaCl, 20 mM HEPES 7.3) was mixed with 12 µg TfpL190B conjugate in 160 µl HBS, after 30 minutes 20 µl adenovirus d1312 and the mixture was added to K562 cells. b) A solution of 800 ng of streptavidin-polylysine in 160 µl of HBS is mixed with 20 µl of biotinylated adenovirus, prepared as described under a), after 30 minutes a solution of 6 µg of pCMVL-DNA in 160 µl of HBS is added and after a further 30 minute solution was mixed with 10 µg TfpL190B conjugate in 160 µl HBS. after 30 minutes it is added to the cells.

c) DNA complexes were prepared in the same way as in b) with the difference that instead of the TfpL190B conjugate a solution of 3.5 µg of poly(L)lysine, p(Lys)290 was added.

Cells are incubated at 37ºC for 24 hours and then harvested for the Lucerase assay. The values shown in Figure 30 represent the total luciferase activity of the transferred cells.

Example 20 - Gene delivery to original bone marrow cells.

Bone marrow stem cells were harvested from mice by inoculating the culture medium (IMDM containing 10% FCS, 5x10-5 M β-mercaptoethanol, 1% IL-3 adapted medium and antibiotics) with an injection needle, (0.4 mm or 0.5 mm diameter) attached to a 1 ml syringe, through isolated femurs and tibias. The cells are then washed once in the culture medium by centrifugation at 100xg, 8 minutes. After that, the cells are resuspended at a concentration of 107 cells/ml and planted in culture flasks. After 4 hours, non-adherent cells are transferred to new T25 flasks and cultured overnight in the presence of 50 µM desferrioxamine.

b) Creation of adenovirus-DNA-transferrin complex.

To form the adenovirus-DNA-transferrin complex, 50 µl of biotinylated adenovirus is incubated with streptavidin-modified polylysine in 20 µl of HBS for 20 minutes. Then 20 µl of HBS containing 6 µg of pCMVL is added. After an incubation period of 20 minutes, 7 µg of mouse transferrin-polylysine conjugate (mTfpL) was added to 160 µl of HBS and the entire mixture was incubated for a further 20 minutes.

c) Transfection

For transfection, bone marrow cells are obtained from the culture medium of T25 flasks by centrifugation at 100xg for 8 minutes. The cell pellet is resuspended in 3 ml of substrate containing 2% FCS and 250 µl of the adenovirus-DNA-transferrin complex, and spread in a new T25 bottle, for 3 hours at 37ºC. Then 3 ml is added and after 2 hours a further 6 ml of substrate containing 10% FCS is added.

d) Determination of luciferase expression.

Bone marrow cells were harvested 48 hours after transfection and analyzed for luciferase expression as described above. Transfection led to the expression of luciferase activity, which corresponds to 310x103 l.j. (light units) per 100 µg of total cellular protein.

Example 21 - Transfection of a neuroblastoma cell with a 48 kb cosmid in the presence of adenovirus.

a) Preparation of a cosmid containing a luciferase-tagged sequence.

A 3.0 kb Sal I fragment, containing a single P. pyralis luciferase tag sequence under the control of the RSV promoter (De Wet et al., 1987), was ligated to the unique Sal I site of cosmid clone Cl-7aAl, to generate concatamers. (Cl-7aAl comprises a 37 kb human genomic DNA fragment of Sau 3A, which does not clearly tag genes, cloned into the Bam HI position of Stratagen's cosmid vector pWE15). The product of the ligation reaction was then removed in vitro and an aliquot of the resulting phage particles infected into E. coli NM544 and plated on LB amp plates. Recombinants were screened by colony hybridization, using a 3.0 kb Sal I fragment, as a hybridization check and array of facts analyzed by restriction labeling. A cosmid assembly (CosLuc) containing a single copy of the Sal I insert was grown and purified on a CsCl gradient (total size = 48 kb).

The small control cosmid pWELuc (12kb) was prepared by digesting CosLuc with Not I, religating, transforming the bacteria and isolating a clone containing the corresponding plasmid. This resulted in the missing human DNA insert and part of the CosLuc.a polylinker in the 12 kb DNA molecule.

b) Cosmid release into neuroblastoma cells.

Cells of the Neuroblastoma line, designated GI-ME-N (Donti et al., 1988) (1 x 106 cells per 6 cm dish) covered with 1 ml of DMEM +2% FCS, were incubated with TfpL/DNA complexes prepared as was described in the chapter "Materials and methods", which contained the indicated amounts of hTfpL, free pL and DNA. As shown, the cell incubation mixtures contained, in addition, either 10 µM chloroquine (columns 3 and 4) or 10 µl adenovirus d1312 containing 5 x 10 11 particles per ml (columns 5 and 6). The last two samples (labeled StpL/Biotin) containing ul of biotinylated adenovirus d1312 (1 x 10 11 particles) were incubated with streptovidin-polylysine (0.8 µg prepared as in Example 19) for 30 minutes, in 150 µl HBS. Then 6 µg DNA (in 150 µl HBS) is added to the sample for 30 min at room temperature, then another 150 µl HBS) is added to the sample for 30 min at room temperature, then another 150 µl HBS containing 6 µg hTfpL + 1 µg free pl. After a further 30 minutes of incubation at room temperature, the mixture was added to the cells. After 2 hours of incubation at 37 ºC, add 4 ml of DMEM + 10% FCS to each dish. 24 hours later cells are harvested and luciferase activity is measured. Figure 31 shows the results.

Example 22 - Gene transfer into original "airway" epithelial cells with the use of conjugate molecular vectors.

Initial studies to evaluate the possibility of using gene transfer with the use of molecular vector conjugates for genetic correction of cystic fibrosis have shown that non-dead cell lines, derived from human airway epithelium, express sensitivity to this method of gene transfer. In order to rule out the possibility that this phenomenon is the result of immortalization of changes in the respiratory epithelium, the transferrin-polylysine molecular conjugate is also evaluated in cells of the original human respiratory epithelium (1ºAE).

1ºAE cells were obtained from patient nasal polyp samples as described by Yankaskas, J.R. et al., 1987. Briefly, tissues are washed with sterile saline, then with Joklik's "Minimal Essential Medium" (MEM) + antibiotics (penicillin 50 µg/ml, streptomycin 50 µg/ml, gentamicin 40 µg/ml) and transferred to the laboratory at a temperature of 4ºC. Cartilage and excess submucosal tissue are removed, and epithelial sheets are incubated with protease solution (Sigma, type 14, 0.1 mg/dl) in MEM at 4ºC for 16 to 18 hours (Wu, R. 1985). Fetal bovine serum (FBS, 10%) was added to neutralize the protease and the cells were detached by gentle mixing. The resulting suspension is filtered through 10 µm nylon mesh to remove debris, then pelleted by centrifugation (150 x g, 5 min) and washed in F12 + 10% FBS.

1ºAE cells are then treated with a transferrin-polylysine conjugate (hTfpL) containing a luciferase-tagged plasmid (pRSVL) as a reporter gene (L.U.). In this analysis, the original cells showed no sensitivity to this vector expressed by the corresponding non-immobilized cell lines (1ºAE: baseline = 429 ±41; + hTfpL = 543 ±L.U.), probably reflecting the relatively low number of transferrin receptors at 1ºAE.

To exploit an alternative target receptor at 1ºAE, conjugates were designed to incorporate a recovery-incompetent adenovirus as part of the ligand (bAdpL; see Example 19a and 19b) to prepare biotinylated adenovirus and streptavidin-polylysine conjugates. A plasmid labeled with luciferase was used as a reporter gene). Human 1ºAE treated with this conjugate shows levels of expression of the reporter gene significantly stronger than the background (+ bAdpL = 2585753 ±453585 L.U.). In addition, 1ºAE derived from other species also shows a high level of sensitivity to gene transfer by this route (mouse = 3230244 ±343153; monkey = 5348880 ±869481 L.U.). Therefore, the ability to perform gene transfer up to 1ºAE establishes the potential utility of this approach for achieving direct “in vivo” gene delivery.

Example 23 - Gene transfer to hepatocytes with molecular conjugate vectors

a) Transfection of tissue culture cells.

Cells of the mouse embryo hepatocyte line BNL CL.2 (ATCC TIB 73) were cultured as described in Example 6. HeLa cells and hepatocytes were cultured in 6 cm plastic petri dishes. Transfection was performed with a cell density of about 3 x 105 cells per dish. Before transfection, the standard medium was replaced with 1 ml of fresh medium containing 2% FCS.

b) Creation of binary complexes.

Biotinylated adenoviruses (approx. 109 PFU; see Example 19 a) and 19 b) are reacted with 800 ng of streptavidinylated polylysine in 50 µl of HBS. After 30 min at room temperature, 6 µg pCMVL-DNA in 170 µl HBS was added, incubated for 30 min and then 3 µg polylysine pL300 in 200 µl HBS was added, and then, after a further 30 min, the solution was used for transfection experiments.

c) Creation of ternary complexes.

Ternary DNA complexes, containing adenovirus and transferrin, were created as follows: biotinylated adenoviruses (about 109 viral particles) were mixed with 800 ng of streptavidinylated polylysine. After 30 min at room temperature, the solution is mixed with 6 µg of plasmid DNA in 170 ml of HBS, incubated for 30 min, then 10 µg of transferrin-polylysine TfpL190B (Wagner, E. et al., 1991b) in 200 µl of HBS is added, and after a further 30 minutes the solution is used for transfection experiments.

d) β-galactosidase experiments

BNL CL.2 cells were plated on coverslips and 24 hours later the cells were transfected with the pCMV-β-gal (Lim, K. and Chae, 1989) reporter gene. After 48 hours, β-galactosidase was tested according to Lim and Chae.

Selected portable complexes. Biotinylated adenovirus was fused to streptovidinylated polylysine. Alternatively, adenovirus is linked covalently to polylysine by the action of transglutaminase. Adenovirus-polylysine conjugate is added to DNA, enabling the formation of a complex between DNA and polylysine, and thus neutralizing a known fraction (approx. 1/4) of the negative charges of DNA. A calculated amount of polylysine was then added to neutralize the remaining charges. Complexes consisting of DNA bound to an adenovirus-polylysine conjugate and to polylysine have been reported as binary transport complexes.

There are essentially two ways to assemble binary transmission complexes. DNA can be bound to strepatvinylated polylysine and then attached to biotinylated adenovirus, or adenovirus is bound to polylysine, and later complexed with DNA. The latter procedure, quite clearly, gives better results, especially with a low DNA content, and is therefore the preferred method for assembling both binary and ternary complexes.

Ternary transfer complexes containing transferrin.

Adenovirus-polylysine conjugates are added to DNA, enabling complex formation and neutralization of part (about 1/4) of the negative charges of DNA. The calculated amount of transferrin-polylysine conjugate is then added to the complex and neutralizes the rest of the DNA. Complexes containing DNA, adenovirus-polylysine and transferrin-polylysine are described as ternary complexes. Essentially, such ternary complexes should have the capacity to be endocytosed by binding to either cellular adenovirus receptors or transferrin receptors.

The association between DNA condensate and adenovirus significantly enhances the expression of the luciferase reporter gene. Physical association between adenovirus, strain d1312, and polylysine can be achieved either by incubating the two components with transglutaminase, or by biotinylation of adenovirus and streptavidinylation of polylysine. The effect of ligation on transfection efficiency (this term denotes transferrin-directed transfection) in the presence of free adenovirus increased, showing a typical enhancement of the release of transferrin-polylysine DNA complexes into cells. At a specific site in pLAdenoV/TpfL, the adenovirus is conjugated to polylysine by transflutaminase and then reacts with DNA neutralizing some of the negative charges on the DNA. Later, transferrin-polylysine is added, which neutralizes the remaining charge. In this way, the training complex adenovirus-polylysine/transferrin-polylysine/DNA is synthesized. As can be seen, the remarkably high value of 1.5 x 109 l.j. (light units) of luciferase is achieved (or approx. 5000 l.j. per cell).

In order to demonstrate the specificity of transglutaminase-directed binding of polylysine to the virus, the enzyme is omitted. The viral preparation is then complexed with the same amount of DNA and TfpL as in pLAdenoV/TfpL. In this case, the transfection was moderate, as in AdenoV + TfpL, because in both experiments the shared location of the virus and transferrin DNA is a stochastic process, in contrast to PLAdenoV/TfpL where co-internalization ensured by the physical connection of virus and DNA and the ternary complex gives a high level of transferinfection.

Transfection of K562 cells reveals endosomolytic properties of adenovirus. The human erythroleukemic cell line K562 contains approximately 150,000 transferrin receptors (Klausner, R.D., et al., 1983b). In the presence of chloroquine, as previously reported by Cotton M. et al., 1990, these cells can be highly transfected with polylysine-transferrin reporter DNA complexes, even in the absence of adenovirus (TfpL, Figure 33). The same complexes supplemented with free adenovirus, and in the absence of corokin, gave relatively weak levels of expression of the reporter gene (AdenoV/TfpL), probably because K562 cells, like other blood cells, (Silver, L. et al., 1988; Horvath , J. et al., 1988), have low levels of adenovirus receptors. When the adenovirus binds to the polylysine via a biotic/streptavidin bridge, and the reporter DNA is completely condensed by the addition of more polylysine to fill the binary complex, (pLAdenoV/pL), adenovirus-assisted transfection reaches intermediate levels. It is likely that some adenovirus receptors on K562 cells are used effectively. If, however, the bound adenovirus-polylysine-reporter DNA is completely condensed and neutralized by the addition of polylysine-transferrin, in order to form a ternary pLAdenoV/TfpL complex and for numerous cellular transferrin receptors to play a role, the efficiency of transfection, thanks to both the efficient binding of transferrin and and endosomolytic properties of the virus, increases at least by two orders of magnitude (Figure 33).

Ternary DNA complexes lead to reporter gene expression in almost 100% of hepatocytes. The efficiency of the new DNA transport complexes was also tested in mouse hepatocytes (BNL CL.2), by determining the percentages of cells that can be reached by our various transfection protocols. The β-galactosidase gene, driven by the CMV promoter, was used as a reporter gene. After the cells have settled, the activity of galactosidase is established according to Lim and Chae, 1989.

Figure 34 shows a galactosidase experiment on mouse hepatocytes after a) transfer infection in the presence of chloroquine, b) transfer infection in the presence of free d1312 adenovirus and c) transfection with ternary, linked (d1312) adenovirus-polylysine-transferrin-reporter DNA complexes. In the absence of adenovirus, after standard reporter-DNA transfer infection, only a few cells express the reporter gene. The percentage of transfection is less than 0.1%. When chloroquine is included, the percentage increases to about 0.2% (Figure 34a). With free adenovirus, about 5 to 10% of cells express the reporter gene (Figure 34b), while ternary complexes with transglutaminase-modified virus lead to expression in most, if not all, cells (Figure 34c). Since ternary complexes can be used in high dilution, the toxic effect, which is noticeable with high doses of adenovirus (free, inactivated) usually does not appear. However, it should be emphasized that in the case when ternary complexes appear in high concentration with the aim of reaching 100% of tissue culture cells, a similar tolstic effect becomes noticeable. Toxic effects may be caused by residual viral activity of the gene, endosomolytic properties of the added virus, or simply as a result of very high expression levels of the transfected gene.

The expression of the transfected reporter gene is transient, but lasts for weeks in non-dividing hepatocytes. Ternary transport complexes (pLAdenoV/TfpL) were made with d1312 polylysine adenovirus and d1312 modified adenovirus, then they were inactivated by reacting the virus with psoralen. Two-thirds of confluent hepatocytic cell cultures were transfected as in Figure 34b, with the luciferase reporter gene CMVL, and luciferase activity was determined at different time points. As can be seen from Figure 35, the luciferase activity is highest after three days, when the hepatocytic cell culture becomes clumped together and the cells stop dividing. Expression of the reporter gene remains in undivided culture cells, without the application of selection for gene maintenance, and lasts at least 6 weeks, especially when proralen-inactivated adenovirus is used to create ternary transferrin transport complexes.

Example 24 - Use of chicken adenovirus CELO to increase DNA delivery to human cells.

In this example, chicken adenovirus CELO was tested for its ability to enhance DNA transfer into human HeLa cells, in a manner similar to the above experiments using human adenovirus 5.

Chicken adenovirus CELO (Phelps strain, serotype FAV-1.7, chicken kidney cell passage) was used in these experiments. Virus (2 ml) is passed through a gel filtration column, prepared with 20 mM HEPES pH 7.3 150 mM NaCl, (HBS) + 10% glycerol and 2 ml eluent is reacted with 20 µl l mM NHS-LC-biotin (Pierce ), 3 hours at room temperature. Biotinylated virus is then dialyzed against 3 x 300 ml HBS + 40% glycerol at 40ºC and aliquots are stored at -70ºC.

HeLa cells (5x105 cells per 6 cm dish) are incubated in 2 ml DMEM + 2% FCS with 6 µg plasmid pCMVL complexed with polylysine (pLys) or transferrin-polylysine (TfpL) mixtures in 500 µl HBS (the complexes were pre-incubated for 30 minutes at room temperature). Samples are then added to the cells at 37ºC in the presence of the amount of virus indicated in Figure 36. With samples containing biotinylated CELO virus, the indicated amount of streptavidin-polylysine (StrpL) in 200 µl HBS is pre-incubated for 30 minutes at room temperature, before the addition of 6 µl plasmid. (pCLuc) in 100 µl HBS. After 30 minutes of incubation at room temperature, the indicated amount of TfpL material was added to the cells at 37ºC. Two hours later, 5 ml of DMEM + 10% FCS was added, and 24 hours later the cells were harvested and prepared for the luciferase experiment. The results are shown in Figure 36.

As seen in Figure 36, CELO virus as a free entity enhances DNA transfer into HeLa cells (columns 1-6). However, when CELO virus was modified with biotin and complexed with streptavidin, either with or without additional transferrin-polylysine, the virus was found to enhance DNA transfer to a level comparable to that achieved using human adenovirus d1312. Some HeLa cell lines show a high capacity to associate with polylysine/DNA complexes in the absence of transferrin (compare the luciferase activity of samples 1 and 4 in Figure 36), therefore inclusion of the CELO virus in the polylysine/DNA complex is sufficient to induce viral uptake.

Example 25 - Transfection of myoblasts.

a) Transfection of myoblasts and myotubes with DNA/transferrin-polylysine complexes in the presence of free adenovirus and in the presence of biotin/streptavidin bound adenovirus

C2Cl2 myoblasts (Blau et al., 1985; ACTT No.: CRL 1772) and G8 myoblasts (ATCC No.: 1456) were cultured in high glucose DMEM + 10% FCS. Myoblast cultures were transferred with approximately 5 x 105 cells per dish. Myotube cultures were prepared by plating myoblasts on 6 cm dishes (about 5 x 105 cells per dish) and changing the medium to high-glucose DMEM + 2% horse serum when the cells reached confluence (Barr and Leiden, 1991; Dhawan et al., 1991). . Transfection of myotubes is performed 5-7 days later. Transfection complexes were prepared as described in Example 19, using the indicated amounts of TfpL, StrpL, and biotinylated adenovirus d1312. Cells were harvested 20 hours after transfection and luciferase activity was measured. Figure 37 shows the obtained luciferase activities in l.j. (Light Units) for the entire cell sample. Myoblast cultures as well as myotube cultures can be transfected very efficiently. Upon differentiation to myotubes, there was less than one log reduction in transfection efficiency (C2Cl2), or no significant reduction (G8). The proportion of myotube formation was observed at lower densities with the G8 line, which can be attributed to the lack of a noticeable decrease in efficiency in the differentiated culture. The role of the transferrin/transferrin receptor interaction in the transfer of DNA in that cell type is not great. In all four cell preparations, there was weak transfer of DNA by TfpL/DNA complexes, in the presence of free adenovirus d1312 (columns 1, 4, 7, 10). Transfection efficiency was enhanced by the use of pooled viral systems (columns 2, 3, 5, 6, 8, 9, 11, 12). There was less than a 1 log increase in efficiency comparing transfer with combination complexes containing only virus and polylysine/StrpL fused to complexes comprising transferrin-polylysine (compare, for example, column 2, no transferrin, with column 3, transferrin ). Low transfection with free virus and high transfection with attached viral complexes, either in the presence or absence of transferrin-pL, suggests that adenovirus serves as a ligand in these cells and in the absence of attachment free virus can enter cells but the TfpL/DNA complex does not. (The DNA used in this example was pCMVL, labeled pCLuc in the figure).

b) Histochemical analysis of transfection frequency in myotubes.

C2Cl2 cultures of myotubes (5 x 105 cells, as myoblasts, seeded per 6 cm dish and differentiated into myotubes) were prepared in a). With free virus samples, pCMV-β-gal DNA (6 µg) is complexed with 8 µg TfpL in 500 µl HBS and added to the cells in the presence of 18 µl adenovirus d1312 (1 x 1012 virus/ml) in 2 ml DMEM/2% FCS . Pooled virus samples are prepared with pCMVLacZ DNA (6 µg) complexed with 7 µg TfpL and 800 ng StrpL + 18 µl biotinylated adenovirus d 1312 (1 x 1012 virus per ml) in 500 µl HBS and added to cells in 2 ml DMEM/2 % FCS. After 24 hours of incubation, the cells were prepared for β-galactosidase activity, as described in example 15. Samples labeled for β-galactosidase were in accordance with the results of transfection with luciferase as a reporter gene (see a). Very weak gene expression was achieved in myotube cultures with free virus, while fusion of virus and DNA led to high levels of gene expression. The presence of bluish multi-nuclear tubules indicated successful gene transfer by these differentiated cells, in the presence of free adenovirus.

c) DNA transfer to mouse primary myoblast and myotube cultures.

The larger skeletal muscles of the hind legs of a 4-week-old male mouse (C5781/6) were sterilely isolated in PBS and ground into pieces of approx. 5 mm. The tissue was suspended in 20 ml of PBS, allowed to settle for about 2 minutes and the supernatant was aspirated. This washing was repeated three times. The tissue was then mixed with 3.5 ml PBS + 0.5 ml trypsin/EDTA, 0.5 ml 1% (wt) collagenase (type II, Sigma), and 0.5 ml 1% BSA (fraction V, in 4 mM CaCl2) and was left to incubate at 37ºC for 30 min, with gentle stirring more often. After 30 minutes of incubation, the remaining tissue is allowed to settle and the supernatant is removed and mixed with 5 ml of DMEM + 20% FCS. Incubation with protease is repeated 3-4 times, until the tissue is completely dispersed. The cell suspension is then passed through a cell strainer (Falcon) to remove clumps and tissue fragments and centrifuged at 500 g for 15 minutes. The cell pellet is resuspended in 10 ml DMEM + 20% FCS and fibroblasts are removed by spreading the cells onto an uncovered 15 cm diameter tissue culture dish for 60 minutes. Free cells are then carefully removed and spread on 5 laminin-coated tissue culture dishes, 10 cm Ø, with 15 ml of DMEM + 20 FCS per dish. Once the cells are confluent (after approximately one week), they are trypsinized and re-plated onto laminin-coated 6 cm dishes, approximately 1 x 106 cells per dish. In order to create myotube cultures, approximately 5 days later (when the cells reach confluence), the medium is changed to DMEM + 2% horse serum, and one week later transfection is performed in 6 cm dishes with approx. 80% confluence of cells. Laminin-coated cell culture plates were prepared as follows. Cell culture dishes are coated with 0.025 mg/ml polylysine (m.wt. 30,000 to 70,000, Sigma) in sterile water for 30 minutes at room temperature. The plates are washed three times with sterile water and air-dried. The plates are then coated with 8 µg/ml laminin (EHS, Sigma) in water overnight at room temperature. The plates are then washed three times with sterile water, before plating the cells.

DNA complexes taken for transfection were prepared by diluting the specified amount of psoralen or UV-irradiation inactivated biotinylated adenovirus d1312 (prepared as described in example 19) in 150 µl HBS and adding 1 µg of StrpL in 150 µl HBS, followed by incubation at room temperature 30 minute. Then, HBS (100 µl) containing 6 µg of pCMVL (marked in the figure with pCLuc) is added to each sample, and incubation is carried out for another 30 minutes at room temperature. Finally, 7 µg of TfpL in 100 µl of HBS is added to each sample incubated for 30 minutes, and added to either myoblast culture or myotube culture, in 6 cm dishes, containing 2 ml of DMEM + 2% FCS. After one hour of incubation, the medium was replaced with 5 ml of DMEM + 20% FCS (myoblasts) or DMEM + 2% horse serum (myotubes) and 48 hours later cells were harvested for luciferase analysis. Luciferase activity from the whole cell sample is shown in Figure 38.

Example 26 - Improvement of CELO virus transition into myoblasts using lectin ligands

a) Comparative analysis of adenovirus d1312 and CELO virus in HeLa and C2Cl2 myoblasts,

Samples of either HeLa cells or C2Cl2 myoblasts (5 x 105 cells per 6 cm dish) were transfected with 6 µg of pCML (marked in the figure as pCluc) complexed with 1 µg of StrpL/7 µg of TfpL + 5 µl of biotinylated adenovirus d1312 (see Example 19 , 1 x 1012 particles/ml) or 18 µl of biotinylated CELO virus (see Example 24, 0.3 x 1012 particles per ml. After 20 hours of incubation, cells are harvested and prepared for measurement of luciferase activity. The results of luciferase activity of each whole cell sample are shown picture 39.

Transfection in HeLa cells can be performed with comparable effects using adenovirus d1312/StrpL/TfpL/DNA complexes, which can enter cells via adenoviral receptors or via transferrin receptors, or using virus/StrL/TfpL/DNA complexes, which can enter via transferrin. receptors. However, while the transfer of DNA into C2Cl2 myoblasts can be efficiently carried out with adenovirus d1312 complexes, complexes containing CELO virus work weakly in these cells. Previous experiments have shown that the transferrin receptor plays a small role in the transition of the combination complex into the cells. It is likely that the adenovirus receptor is the main site of entry. The weak activity of CELO virus in myoblast may, then, be caused by the weak binding of both CELO virus and transferria to C2C12 myoblasts.

b) Improvement of transfection of C2Cl2 myoblasts with CELO virus, using wheat germ agglutinin as a ligand.

Due to the free transition achieved in a), a new ligand was chosen as a substitute for transferrin.

Biotinylated wheat germ alutinin (2–4 mol biotin per mol protein) was purchased from Boerhringer Mannheim. Biotinylated CELO virus was prepared as previously described. The complexes, which contain 6 µg of pCMVL + indicated amounts of StrpL, TfpL, biotinylated wheat germ agglutinin (WGA-B) and CELO virus, were prepared as follows. Virus and WGA were diluted together in 150 µl HBS and both solutions were mixed and incubated at room temperature for 30 minutes. DNA diluted in 100 µl HBS is added to the StrL/Virus/WGA solution and incubation is carried out for a further 30 minutes at room temperature. Finally, TfpL in 100 µl HBS was added and the mixture was incubated for another 30 min at room temperature. The complexes are added to C2Cl2 myoblasts (5 x 105 cells per 6 cm dish) in 2 ml DMEM + 2% FCS. One hour later, 5 ml of DMEM + 10% FCS was added to the cells, and 20 hours later the cells were prepared for measuring luciferase activity. The activity (light units) in each whole cell sample is shown in Figure 40. (The DNA used in this example was PCMVL, labeled in the figure as pCluc).

In the absence of virus, very weak DNA transfer was achieved, either with or without WGA (columns 1, 6). Moderate transition was achieved with the fused CELO virus (column 2), however, if WGA was included in the complex, a 16-fold increase in transition was achieved. Increasing the amount of WGA in the complex (from 1 µg to 5 µg) led to a slight decrease in the transition (compare columns 3 and 4), while increasing the content of StrpL in the complex (from 1 µg to 2 µg) slightly increased the transition (compare columns 3 and 5 ). These results clearly demonstrate that WGA-B as a ligand increases CELO virus-driven DNA transfer into C2Cl2 cells.

d) Expression of the full-length factor VIII gene in C2Cl2 myoblast and myotube cultures.

Cultures of C2Cl2 myoblasts and myotubes were prepared as described above. Transfections were performed with 6 µg of plasmid encoding full-length human factor VIII cDNA (Wood et al., 1984; Eaton et al., 1986) complexed with 5 or 15 µl of biotinylated adenovirus (as indicated) + 0.5 or 1 µg StrpL, and 7 or 6 µg of TfpL in a standard complexation protocol.

DNA/virus complexes were added to the cells in 2% FCS/DMEM. After 4 hours of incubation at 37ºC, add 3 ml of fresh DMEM + 10% FCS to each dish. After 18 hours, the substrate is harvested and a test for the presence of factor VIII is carried out, using the COATEST (KABI, Pharmacia) test system with an international standard as a reference sample. Factor VIII is entered in the diagram in m units created in 24 hours per 1 x 106 cells (Figure 41).

Example 27 - Use of adenovirus protein for DNA transfer.

Adenovirus wt300 was grown in HeLa cells, purified and biotinylated as described for adenovirus d1312. 1.2 ml of virus is dialyzed against 3 x 300 ml of 5 mM MES, 1 mM EDTA pH 6.25 4ºC, 18 hours. The material was then centrifuged for 30 minutes at 27 K in a SW60 rotor. The supernatant was carefully removed, the pellet was resuspended in HBS/40% glycerol. HEPES pH 7.4 and NaCl were added to the supernatant to 20 mM and 150 mM, and then parts of the pellet (containing the viral core and the hexon capsid mass, the “core” in Figure 42) as well as the supernatant were tested for DNA transfer into Mov13 mouse fibroblasts as well (Strauss and Jaenisch, 1992) and in HeLa cells.

The creation of a complex with DNA was carried out in the following way. Indicated amounts of each fraction, lysed virus prior to centrifugation or intact virus (expressed in µg protein as determined by Bradford), were diluted in 300 µl HBS). Streptavidin-polylysine (3 µg in 50 µl HBS) was added and then incubated for 30 min at room temperature. 6 µg of pCMVL (labeled pCluc in the image) was diluted in 100 µl of HBS and added to the first solution for incubation for 30 minutes. Samples prepared with only TflpL in 170 µl HBS were mixed with 6 µg pCMVL in 330 µl HBS for 30 min at room temperature. The indicated amounts of viral protein are diluted in 300 µl HBS and then added to the TfpL/DNA complexes. All samples were then added to 5 x 10 5 cells, in a 6 cm dish, containing 2 ml DMEM/10% FCS (either HeLa or Mov13 fibroblasts), for 1 hour. Then 5 ml of fresh medium containing 10% FCS is added and the cells are prepared for luciferase activity 20 hours later. The result of luciferase activity (in light units) is shown in Figure 42, for HeLa cells (graph A) or for Mov13 fibroblasts (graph B).

In both groups of cells, there is a dose-dependent activity of DNA translation, related to the highest fraction (sample 4-6 in both graphs). When the same amount of biotinylated viral protein was encapsulated with TfpL/DNA complexes, without streptavidin-polylysine, a DNA transition was observed quite at baseline levels (sample 3 in each graph).

Example 28 - Enhanced gene transfer using DNA ternary complexes containing a galactose ligand conjugate.

a) Ternary complexes containing influenza peptide conjugate.

The presence of polylysine-conjugated peptides, which contain sequences derived from the N-terminus of the influenza viral hemagglutinin, subunit HA-2, in DNA/transferrin-polylysine complexes significantly increases gene transfer controlled by transferrin-polylysine. (Examples 13 and 14).

Similar combinatorial DNA complexes, containing a "tetra-antenna" ligand-polylysine conjugate of galactose and the polylysine-modified influenza peptide InflupL (prepared as described in Example 6 and 13, respectively), were prepared by adding the ligand-polylysine conjugate to plasmid DNA pCMVL, to neutralizes half of the DNA charge, with the rest of the charge being used to charge the complex with the influenza conjugate. The transfection of these DNA complexes, which contain the synthetic ligand (gal)4, with BNLCL.2 hepatocytes (transfections were performed as described in example 6 g), resulted in the expression of the luciferase gene (Figure 43) which is significantly higher than the expression achieved with transferrin as a ligand. The expression is more than 500 times higher than in control experiments, achieved with DNA complexes without influenza peptide, but with the same amount of polylysine (Figure 43). The activity achieved with DNA combination complexes was also about 30 times higher than with DNA/(gal)4pL complexes incubated with cells in the presence of chloroquine.

b) Ternary complexes containing adenovirus conjugate.

Complexes were prepared as follows: biotinylated adenovirus d1312 (prepared as in Example 19; 2 µl, 6 µl or 18 µl; 1012 particles/ml) in 50 µl HBS, was mixed with streptavidin-polylysine (100 ng, 160 ng or 480 ng ) in 100 µl hbs. After 30 min of incubation, a solution of 6 µg of pCMV-L in 200 µl of HBS was added and, after a further 30 min, a solution of 3.8 µg of (gal)4pL (prepared as in Example 6) or 7 µg of TfpL in 150 µl was added. HBS.

The DNA complex solution is added to every 300,0000 cells (ATCC TIB 73, ATCC TIB74, ATCC TIB75, ATCC TIB76) grown on 6 cm plates in high glucose DMEM + 2% FCS. Further cell culture experiments and luciferase assays were performed as described. Gene expression (after 24 hours) is shown in Figure 44.

Example 29 - Transfer of DNA with transferrin-polylysine in the presence of free and conjugated rhinovirus

a) Preparation of rhinovirus HRV-2.

Rhinovirus HRV-2 was prepared and purified as described (Skern et al., 1984).

400 µl of rhinovirus solution (approx. 30 µg) in HBS (150 mM NaCl/5 mM HEPES pH 7.9)/10% glycerol, was treated with 10 nmol NHS-LC-biotin (Pierce 21335). After 3 hours of incubation at room temperature, the virus was separated from unincorporated biotin by long dialysis against HBS/glycerol at 4ºC.

Light-sensitive rhinovirus, prepared by growing the virus in the presence of acritin orange, was inactivated as described (Madshus et al., 1984).

b) Preparation of DNA complex and transfection.

I) Transferrin-polylysine / DNA complexes were prepared by mixing a solution of 6 µg DNA plasmid pCMVL and 330 µl HBS (150 mM NaCl, 20 mM HEPES, pH 7.3) with a solution of 8 µg TfpL290 in 170 µl HBS.

DNA complexes are mixed with 1.5 ml of substrate (DMEM + 2% FCS) and with 0.14 µg to 3.5 µg of rhinovirus HRV-2 (or inactivated HRV-2). The mixture is added to NIH 3T3 cells (300,000 cells per 6 cm plate). Four hours later, the transfected medium was replaced with 4 ml of fresh DMEM + 10% FCS. Cells were harvested after 24 hours and luciferase activity was measured as previously described (Figure 45A).

II) DNA combination complexes, which contain transferrin-polylysine and rhinovirus-polylysine conjugates, were prepared as follows:

100 µl of a solution of biotinylated rhinovirus HRV-2 (3.5 µg) in HBS was mixed with 1 µg of streptavidinylated polylysine in 100 µl of HBS. (other combinations of viruses are mixed with appropriate amounts). After 30 min at room temperature, the solution was mixed with 6 µg of plasmid DNA in 150 µl of HBS, incubated for a further 30 min at room temperature and then mixed with 6 µg of TfpL290 in 150 µl of HBS.

DNA complexes are mixed with 1.5 ml medium (DMEM + 2% FCS) and added to NIH 3T3 cells (300,000 cells per 6 cm plate).

Further processing of cultures and assay for luciferase activity was performed as described in I) (Fig. 45B).

Example 30 - Transfection of HeLa cells with combination complexes containing ionically bound adenovirus

Complex formation A) DNA complexes are prepared by mixing 30 µl of adenovirus d1312 (approx. 109 PFUs) with 1 µg of polylysine pLys450 (average chain length of 450 monomers) in 170 µl of HBS and, after 30 min at room temperature, mixing with 6 µg of pCMVL -DNA in 170 µl HBS. After incubation for a further 30 min, the complexes were mixed with 9 µg of TfpL190 in 170 µl of HBS. An aliquot of the complex mixture (10% = 50 µl solution, 600 ng DNA; or 1% 0 5 µl solution 60 ng DNA) is diluted in 1.5 ml DMEM + 2% FCS and added to 300,000 HeLa cells. After 4 hours, 2 ml of DMEM + 20% FCS is added. 24 hours after transfection, cell harvest and luciferase assay were performed as described. The luciferase activity, corresponding to the total extract, was 29115000 l.j. ("Light units"), in the case of 600 ng DNA and 1090000 l.j. in the case of 60 ng DNA.

Control experiment: Complex formation B) DNA complexes were prepared by first mixing 6 µg pCMVL-DNA in 170 µl HBS with 1 µg polylysine pLys450 (average chain length of 450 monomers) in 170 µl HBS and after 30 minutes at room temperature, mixing with 9 µg TfpL190 in 170 µl HBS. After a further 30 min of incubation, the complexes are mixed with 30 µl of adenovirus d1312 (approx. 109 PFUs). An aliquot of the complex mixture (10% 0 50 µl solution, 600 ng DNA; or 1% = 5 µl solution, 60 ng DNA) is diluted in 1.5 ml DMEM + 2% FCS and added to 300,000 HeLa cells. After 4 hours, 2 ml of DMEM + 20% FCS is added. Cell harvest and luciferase assay were performed 24 hours after transfection, as previously described. The luciferase activity corresponding to the total extract was 405,000 l.j. (in the case of 600 ng DNA) and 200 l.j. (in the case of 60 ng DNA).

Example 31 - Local application of DNA/adenovirus/transferrin-polylysine conjugate in rat liver.

a) Direct injection

The complexes were prepared as described in Example 19. They comprise 200 µl of biotinylated adenovirus d1312, 6.4 µg of streptavidin-polylysine, 48 µg of pCMVL and 48 µg of TfpL290 in a total volume of 2000 µl of HBS. A 240 g male Sprague-Dawley rat was anesthetized with Avertin and a 4 cm laparotomy was performed. The complex solution was injected into the left lobe of the liver. then the wound is closed in layers. After 48 hours of injection of the complex, the rat was sacrificed and the luciferase activity was measured. In the area of injection, 5615 IU/per mg of liver homogenate protein was measured. The total luciferase activity at the injection site was 370,600 l.j.

b) Administration of the conjugate in the liver via the bile drainage system.

Complexes were prepared as follows: 200 µl of biotinylated adenovirus d1312 was diluted with 200 µl of HBS and incubated with 6.4 µg of streptavidin-modified polylysine in 400 µg of pCMVL in 800 µl of HBS. After 30 min of incubation, another 48 µg of TfpL in 900 µl of HBS was added. For the application of the complex, male Sprague-Dawley rats (250 g body weight) were anesthetized with Avertin and the abdomen was opened through a central incision. The intestines were moved to the left side of the body and a G 27 needle was inserted into the bile duct, which was attached to a tube and a 1 ml syringe. The injection of the complex was performed over a period of 4 minutes. Then the needle was withdrawn from the catheter and the injection site was glued with fibrin glue (immuno). The abdominal wound is closed with stitches and metal clips. After 30 hours, the rat was sacrificed and samples from different lobes of the liver were examined for the expression of the luciferase gene. The maximum of luciferase activity was 19,000 l.j./mg protein, and the calculated total expression in the whole liver was in the range of 2.7 x 106 light units.

Example 32 - Local application of DNA/adenovirus/tranferin-polylysine conjugate in a clamped mouse tail vein.

Complexes were prepared as described in Example 19.

They contain 45 µl biotinylated adenovirus d 1312, 0.8 µg streptavidin-polylysine, 6 µg pCMVL and 24 µg TfpL290 in a total volume of 170 µl HBS. The complexes were injected into the tail vein of a male C3H/He mouse (two months old), which was anesthetized with Avertin. Immediately after the injection, the vein was clamped for 20 minutes at the proximal and distal ends of the tail, so that the complex solution was limited to the segment of the tail vein into which it was injected, and could not spread through the blood. 48 hours after the injection, the mouse was sacrificed and the tail vein was dissected. Luciferase expression was measured in the homogenate of the vne tail segment. The expression resulted in 2,600 l.j./3 cm tail.

Example 33 - Transfection of original human melanoma cells.

Primary melanoma cells were isolated from melanoma, which was surgically removed from the patient, by mechanically crushing the tumor in RPMI 1640 medium + 5% FCS, 2 mM glutamine and antibiotics and pressing the tissue fragments through a steel sieve. Tumor cells were washed by centrifugation and resuspended and seeded into T25 vials for cell culture. 24 hours after isolation, tumor cells were transfected with combination complexes containing 3 µl, 9 µl or 27 µl of biotinylated adenovirus d1312 (1 x 1012 virus/ml), 0.5 µg of streptavidin-polylysine, 6 µg of pCMVL and 7 µg of TfpL290 in total. volume of 500 µl HBS. 36 hours after transfection, cells are harvested and luciferase expression is determined. The results are shown in Figure 46.

Example 34 - Transfection of original human fibroblasts.

Human skin biopsies were placed on a 6 cm petri dish containing DMEM, 2 mM glutamine, 20% FCS and antibiotics. Then the biopsies were thoroughly minced with a surgical knife and cultured in the presence of 3 ml of substrate for 5 days. After that, the cells were washed with fresh DMEM, containing 2 mM glutamine, 10% FCS and antibiotics, and cultured for a further 7 days. After that, the cells were trypsinized and grown again in new petri dishes. When the cells were almost confluent, they were trypsinized again and kept frozen until transfection. For transfection, cells are thawed and planted in 6 cm petri dishes and grown in DMEM, which contains 2 mM glutamine, 10% FCS and antibiotics. Transfected conjugates are prepared as follows: 3 µl, 10 µl, 20 µl and 30 µl of biotinylated adenovirus d1312 are incubated with 0.1 µg, 0.3 µg, 0.5 µg and 0.8 µg of polylysine-modified streptavidin in 150 µl HBS , 30 minutes at room temperature. Then, 6 µg of pCMV-gal plasmid was added to 170 µl of HBS and the mixture was incubated for a further 30 minutes. In the final step, 7.8 µg TfpL for conjugates with 3 µl d1312, 7 µg TfpL for 10 µl d1312 and 6 µg TfpL for conjugates with 20 µl and 30 µl d1312 and 179 µg HBS are added. After 30 minutes of incubation, the conjugates are added to the cells in 2 ml of DMEM containing 2 mM glutamine, 2% FCS and antibiotics and the cells are incubated for 4 hours at 37ºC with DMEM containing 2 mM glutamine, 10% FCS and antibiotics. After 48 hours, the expression of β-galactosidase was demonstrated, as described in the previous examples.

In transfection with 3 µl d1312 14% of cells showed β-galactosidase production, with 10 µl d1312 32% of positive cells were obtained, with 20 µl d1312 39% and with 30 µl d1312 64% of cells were positive.

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

a) Synthesis of membrane-disrupting peptides.

I) Peptide synthesis:

Peptides were synthesized on an automatic synthesizer (ABI 431A) by the solid-phase method, using p-alkoxybenzylalcohol resin (0.97 nmol/g) as a solid support and Fmoc-protected amino acids. The carboxy-terminal amino acid is coupled to the resin via 1-hydroxy-benzotriazole-dicyclohexylcarbodiimide. The following side chain protecting groups were used: (Trt)Asn, (Trt)Cys (t-Bu)Cys in the case of EALA and CLF, (t-Bu)Glu, (Trt)His, (t-Bu)Ser.

EALA: (SEQ ID NO:5) Trp Glu Ala Ala Leu Ala Glu Ala Leu Ala Glu Leu Ala Glu His Leu Ala Glu Ala Leu Ala Glu Ala Leu Glu Ala Leu Ala Ala Gly Gly Ser Cys

GLF (SEQ ID NO:6) 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 Cly Ser Cys

GLF-II (SEQ ID No:7) 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 Ser Cys

GLF-delta (SEQ ID No:8) 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-Inf (SEQ ID No:9) 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 (SED ID No:10) 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 Cyd

P50 (SED ID No:11) 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 cleaved from the resin and the protective groups of the side chain (except (t-But)Cys) are removed, by treating 100 mg of the support on which the peptide is, with 3 ml of a mixture of phenol/ethanedithiol/thioanisole/water/trifluoroacetic acid in a ratio of 0.75 : 0.25 : 0.5 : 0.5 : 10, at room temperature for 1.5 h. Crude peptides are precipitated in ether and washed twice. The S-t-Bu protected peptides EALA and GLF were dissolved in a small volume of 1M triethylammonium bicarbonate (TEAB) pH 8, diluted to 100 mM TEAB and further purified by reverse phase high pressure liquid chromatography (HPLC) on a Nucleosil 500-5C4 column (0 ,1% TFA-acetonitrile gradient). Both peptides are eluted with about 50% Ac-nitrile. The free Cys-SH form of the peptide is obtained by deprotection of the TrT-Cys peptide, in the same way as described above. Crude peptides (5 mg) were dissolved in 100 µl 100 mM TEAB, pH 8, containing 1 µl β-marcaptoethanol and purified by gel filtration (Sephadex G-25, 100 mM TEAB, 0.5 mM EDTA) and freeze-drying, or ion -exchange chromatography (Mono Q Pharmacia, 20 mM HEPES, pH 7.3 gradient 0 to 3M NaCl, peptide is eluted with 1.5 M NaCl).

II) Modification with N-(hydroxyethyl)maleimide

The C-terminal SH group of GLF-delta, GLF-II, EALA-Inf, EALA-P50, P50 is blocked after gel filtration of the free SH form (sephadex G-25, 20 mM HEPES, pH 7.3 0.5 mM EDTA) by reaction with a 1.3 - 10-fold molar excess of N-(hydroxyethyl)maleimide (1 h at room temperature). Excess maleimide is removed by gel filtration (Sephadex G-25, 100 mM TEAB, pH 8) and peptide (GLF-delta-mal, GLF-II-mal, EALA-Inf-mal. EALA-P50-mal,, P50-mal ) are obtained as triethylammonium salts, after freeze-drying.

III) Modification with 2,2'-dithiobispiridine:

Free SH peptides are reacted with 10 equivalents of 2,2'-dithiobispyridine (20 mM HEPWS, pH 7.9 0.5 mM EDTA) overnight at room temperature. Excess reagents were removed by gel filtration (Sephadex G-25, 100 mM REAB, pH 8) or ion exchange chromatography (Mono Q Pharmacia, 20 mM HEPES, pH 7.3, gradient 0 to 3 M NaCl, peptide eluted with 1, 5 M NaCl) and (2-pyridylthio)-Cys peptides (GLF-delta-SSPy, GLF-II-SSPy, EALA-P50-SSPy, P50-SSPY) are obtained.

IV) Peptide dimerization:

Peptide monomer 150 (150 dim) was prepared by reacting equimolar amounts of P50-Cys-(2-pyridylthio) and P50-Cys-SH in 20 mM HEPES, pH 7.3, for three days at room temperature. The reaction mixture was separated on a Mono Q column (HR 5/5 Pharmacia; 20 mM HEPES, pH 7.3, gradient 0.09 3 M NaCl, P50-dimer eluted with 1.1 NaCl). The heterodimer GLF-SS-P50 was prepared analogously, by reacting the peptide P50, free mercapto form, with the pyridylthio-modified peptide GLF.

b) Liposome leakage experiment:

The ability of synthetic peptides to disrupt liposomes was investigated by releasing fluprescent dye from liposomes charged with a self-quenching concentration of calcein. Liposomes were prepared from egg phosphatidylcholine by reverse-phase evaporation (Szoka and Papahadjopoulos, 1978) with an aqueous phase of 100 mM calcein, 375 mM Na+, 50 mM NaCl, pH 7.3 and passing through a 100 nm polycarbonate filter (MacDonald et al., 1991). ) to achieve a uniform size distribution. Liposomes were separated from the uniform material by gel filtration on Sephadex G-25, with iso-osmotic buffer (200 mM NaCl, 25 mM Hepes, pH 7.3). For permeation testing at different pH values, a standard liposome solution was diluted (6 µl/ml) in 2x test buffer (400 mM NaCl, 40 mM Na-citrate). An aliquot of 100 µl is added to 80 µl of a serial dilution of the peptide in water, in a 96-well microtiter plate (final lipid concentration: 25 µM) and examined for calcein fluorescence at 600 nm (excitation 490 nm) with a microtiter plate fluorescence photometer. , after 30 minutes of incubation at room temperature. The value for 100% leakage was obtained by adding 1 µl of 10% Triton X-100 solution. Permeation units were calculated as the reciprocal of the peptide concentration, where 50% permeation was observed (i.e., the volume (µl) of the liposomal solution containing up to 50% per µg of peptide. Values below 20 units are extrapolated. The results of liposome permeation experiments are shown in Figure 47 GLF and EALA show the highest pH-specific activities.

c) Erythrocyte disintegration experiment:

Fresh human erythrocytes are washed several times with HBS and resuspended 2x in test buffer of the appropriate pH value (300 mM NaCl, 30 mM Na-citrate) in a concentration of 6.6 x 107/ml. An aliquot of 75 µl is added to 75 µl of a serial dilution of the peptide in water, in a 96-well micro-titre plate (conical type) and incubated for 1 h at 37ºC, with constant shaking. After removal of undegraded erythrocytes by centrifugation (1000 rcf, 5 min), 100 ul of the supernatant is transferred to a new microtiter plate and hemoglobin absorbance at 450 nm (baseline correction at 750 nm) is determined. 100% dissolution was determined by addition of 1 µl of 10% Triton X-100, before centrifugation. Hemolytic units were calculated as the reciprocal of the peptide concentration, when 50% lysis was observed (ie, the volume (µl) of erythrocyte solution that was 50% lysed per µg of peptide). Values below 3 hemolytic units are extrapolated. The values are given in Figure 48. As can be seen, P50 dim and EALA-P50 show the highest pH specific activities with respect to cell lysis and/or release of larger molecules such as hemoglobin. P50 monomers P50mal and P50 SS-Py have lower activity. Melitin showed the highest activity, but this activity is not specific for acidic pH values.

d) Preparation of DNA combination complexes:

DNA complexes were prepared by first mixing 6 µg pCMVL-DNA in 150 µl HBS with 4 µg TfpL290 in 150 µl HBS, and then mixing with 4 to 20 µg poly(L)lysine290 in 100 µl HBS, after 30 minutes at room temperature. After a further 30 min of incubation at room temperature, 0.3 to 30 µg of peptide in 100 µl of HBS is added and incubated for another 30 min. The optimal amount of endosomolytic agent is determined by previous titrations testing the efficiency of gene transfer (see Table 3, for gene transfer in BNLCL.2 pages). The simultaneous addition of plVS and the endosomolytic agent, as well as the use of larger volumes for complex preparation (1.5 ml final volume) gave, as can be seen, comparable (or better) transfection effects. These experiments showed that non-peptide amphipathic compounds, deoxycholic acid and oleic acid, also enhance DNA transfer.

e) Cell transfection:

The respective cell lines (BNL CL.2 hepatocytes or NIH 3T3 cells) were grown in 6 cm dishes for 1 to 2 days before transfection (DMEM medium with 10% FCS; 300,000 cells per dish). The medium is removed and k.5 ml of DMEM (2% FCS) and 500 µl of DNA complex are added. Alternatively, use 0.5 ml DMEM 6% FCS and 1.5 ml DNA complex. After 4 hours of incubation, 2 ml of DMEM (18% FCS) was added.

Alternatively, the transfection medium can be replaced with 4 ml of DMEM with 10% FCS. Cell harvesting and luciferase assays were performed 24 hours after transfection, as previously described. Values of l.j. - (lihgt units) that are shown represent the total luciferase activity of the transfected cells.

Transfection of BNL CL.2 hepatocytes is shown in Figure 49.

Figure 49A) DNA complexes were prepared, first, by mixing 6 µg pCMVL-DNA in 250 µl HBS with 4 µg TfpL290 in 250 µl HBS, and then mixing with 20 µg poly(lysine290 in 750 µl HBS) after 30 minutes at room temperature. After a further 30 min incubation at room temperature, the indicated amounts of peptides in 250 µl HBS were added to the mixture.After a further 30 min incubation, the complexes were mixed with 0.5 ml DMEM + 6% FCS and added to 450,000 cells.

Figure 49B) DNA complexes are prepared as follows: A solution of 6 µg pCMVL-DNA in 500 µl HBS is mixed with 4 µg TfpL290 in 250 µl HBS and left for 30 min at room temperature. A solution of 20 µg poly(L)-lysine in 500 µl HBS is mixed with the indicated amounts of peptide in 250 µl HBS and immediately added to the TfpL/DNA mixture. After a further 30 min of incubation at room temperature, the complexes were mixed with 0.5 ml of DMEM + 6% and added to 450,000 cells. Cells were harvested 24 hours after transfection and luciferase assays were performed as previously described.

Experiments performed with NIH3T3 cells are shown in Figure 50. Complex preparation, according to A9 and B) was the same as for TIB 73 transfection.

In cell culture experiments, p50 dim and ELA P50 showed the highest activity, EALA and GLF medium, while P50 monomers and melittin had weak activity.

Example 36 - Gene transfer using a synthetic non-viral peptide with an oligolysine C-terminal extension.

The peptide sequence (SEQ ID NO:4) 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 Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys, was synthesized and purified according to the method described in the previous example. This peptide is derived from Staphylococcus aureus δ-toxin (SEQ ID NO:3) 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, which is known to possess specificity for membrane disruption at acidic pH (Thiaudiere et al., 1991; Alouf et al., 1989), by extension with an additional 10 lysine residues

DNA complexes were prepared, at the latest, by mixing 6 µg pCMVL-DNA in 170 µl HBS with 4 µg TfpL290 in 170 µl HBS, and then mixing with approximately 3 µg peptide in 170 µl HBS, after 30 minutes at room temperature. After incubation for a further 30 minutes, the complexes are mixed with 1.5 ml of DMEM + 2% FCS and added to 450,000 BNL CL.2 hepatocytes. After 2 hours, 2 ml of DMEM + 20% FCS is added. Harvesting of cells 24 hours after transfection and measurement of luciferase activity is carried out as described in the previous examples. The luciferase activity corresponding to the total extract was 481,000 light units.

Example 37 - Transfection of hepatocytes in the presence of melittin-peptide with a C-terminal oligo-Lys tail.

Sequence peptides (N to C terminus) (SEQ ID NO:12) 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 Lys (marked with mel 1)

and (SEQ ID NO:13) Gly Ile Gly Ala Val Leu Glu Val Leu Glu Thr Gly Leu Pro Ala Leu Ile Serp Trp Ile Lys Arg Lys Ark Gln Gln Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys (acidic mutant, indicated mel 2), synthesized as described in Example 36.

DNA complexes were prepared, first, by mixing 6 µg pCMVL-DNA in 170 µl HBS with 4 µg TfpL290 in 170 µl HBS, and then mixing with approximately 3 µg peptide mel l or 5 µg peptide mel 2 in 170 µl HBS, after 30 minutes. , complexes are mixed with 1.5 ml DMEM + 2% FCS and added to 450,000 BNL CL. 2 cells, grown as described in example 36. After 4 hours, 2 ml of DMEM + 20% FCS are added. Cell harvest, 24 h after transfection and luciferase assays, were performed as described. The luciferase activity, corresponding to the total extract, was 9200 "light units" (in the case of mel l) and 9400 "light units" (in the case of mel 2).

Example 38 - Expression of interferon alpha in HeLa cells.

HeLa cells (5 x 10 5 cells per 6 cm dish) were transfected with pAD-CMV1-IFN encoded by human interferon alfa2c, under the control of the CMV enhancer/promoter (described in DE 40 21 917 A. pADCMV1-IFN was obtained by HindIII-XbaI recloning IFN-2c insert in pADCNV1). Samples of 6 µg DNA in 330 µl HBS were mixed with 8 µg TfpL in 330 µl HBS and allowed to bind for 30 min at room temperature. Samples 6-10 contained only 4 µg of TfpL, and after the first 30 minutes of incubation, an aliquot of P16pL (20 µg) in 160 µl of HBS was added to samples 6 and 7 and an aliquot of pLys 290 (20 µg) to samples 8 and 10. After for a further 30 min of incubation, an aliquot of 160 µl of HBS was added, which contained 10 µl (sample 8) or 50 µl (samples 9 and 10) of free P16 (see example 13 for the synthesis of P16 and P16pL). After an additional 30 minutes of incubation, the samples are added to HeLa cells in 2 ml of DMEM/2%FCS, in the presence of the following additional compounds. Samples 2, 7 and 10 contained 100 µM chloroquine, sample 3 and 4 contained 5 and 15 µl of adenovirus d1312, (1 x 1012 particles/ml), sample 5 contained 15 µl of the same virus inactivated by psoralen. To control adenoviral stimulation of endogenous interferon production, samples 11, 12 and 13 were treated with virus aliquots equal to samples 3, 4 and 5). Two hours after transfection, 5 ml of fresh DMEM + 10% FCS was added. 48 hours after transfection, the medium is removed and replaced with 2 ml of fresh DMEM + 10% FCS. This medium was harvested 72 hours after transfection and an ELISA assay for interferon alpha was performed, as described in DE 40 21 017. Levels of interferon alpha (in ng/ml) are shown in Figure 51. TfpL had a weak effect on IFN gene switching these cells, in agreement with previous observations with luciferase or β-gal reporter genes. The presence of chloroquine produced a detectable signal (approx. 7 ng/ml, sample 2), but adenovirus d1312 stimulated DNA transfer in a dose-dependent manner (samples 3 and 4). Treatment of these cells with comparable amounts of virus, in the absence of the IFN DNA complex, did not result in a detectable interferon signal (samples 11 and 12).

Transfection with the synthetic, influenza-derived endosomolytic peptide P16 (see Example 13), as a conjugate (sample 6, 7) or as an ionically linked peptide to the surface of the TfpL/DNA complex (samples 8, 9 and 10; for peptide binding see Example 35) produced noticeable levels of interferon production, which was increased by the peptide conjugate in the presence of coorokine (sample 7)

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SEQUENCE LISTING

(AND GENERAL INFORMATION:

(ii) TITLE OF INVENTION: Composition for introducing nucleic acid complexes intro higher eukaryotic colls

(iii) NUMBER OF SEQUENCES: 13

(iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0. Version #1.25 (EPO)

(2) INFORMATION FOR SEQ ID NO:1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 amino acids

(B) TYPE: amino acid

(C) STANDEDNESS: single

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptides

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

Gly Leu Phe Glu Ala Ile Ala Gly Phe Ile Glu Asn Gly Trp Glu Gly

1 5 10 15

Met Ile Asp Gly Gly Gly Cys

20

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 amino acids

(B) TYPE: amino acid

(C) STANDEDNESS: single

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptides

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Gly Leu Phe Glu Ala Ile Ala Gly Phe Ile Glu Asn Gly Trp Glu Gly

1 5 10 15

Met Ile Asp Gly Gly Gly Cys

20

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 amino acids

(B) TYPE: amino acid

(C) STANDEDNESS: single

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptides

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

Met Ala Gln Asp Ile Ile Ser Thr Ile Gly Asp Leu Val Lys Tpr Ile

1 5 10 15

Ile Asp Thr Val Asn Lys Phe Thr Lys Lys

20 25

(2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 36 amino acids

(B) TYPE: amino acid

(C) STANDEDNESS: single

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptides

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

Met Ala Gln Asp Ile Ile Ser Thr Ile Gly Asp Leu Val Lys Tpr Ile

1 5 10 15

Ile Asp Thr Val Asn Lys Phe Thr Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys

20 25 30 35

(2) INFORMATION FOR SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 34 amino acids

(B) TYPE: amino acid

(C) STANDEDNESS: single

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptides

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

Trp Glu Ala Ala Leu Ala Glu Ala Leu Ala Glu Ala Leu Ala Glu His

1 5 10 15

Leu Ala Glu Ala Leu Ala Glu Ala Leu Glu Ala Leu Ala Ala GLy Gly Ser Cys

20 25 30

(2) INFORMATION FOR SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 35 amino acids

(B) TYPE: amino acid

(C) STANDEDNESS: single

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptides

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

Gly Leu Phe Gly Ala Leu Ala Glu Ala Leu Ala Glu Ala Leu Ala Glu

1 5 10 15

His Leu Ala Glu Ala Leu Ala Glu Ala Leu Glu Ala Leu Ala Ala Gly Gly Ser Cys

20 25 30 35

(2) INFORMATION FOR SEQ ID NO: 7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 35 amino acids

(B) TYPE: amino acid

(C) STANDEDNESS: single

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptides

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:

Gly Leu Phe Gly Ala Leu Ala Glu Ala Leu Ala Glu Ala Leu Ala Glu

1 5 10 15

Ala Leu Ala Glu Ala Leu Ala Glu Ala Leu Glu Ala Leu Ala Ala Glu Gly Ser Cys

20 25 30 35

(2) INFORMATION FOR SEQ ID NO: 8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 34 amino acids

(B) TYPE: amino acid

(C) STANDEDNESS: single

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptides

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

Gly Leu Phe Gly Leu Ala Glu Ala Leu Ala Glu Ala Leu Ala Glu Ala

1 5 10 15

Leu Ala Glu Ala Leu Ala Glu Ala Leu Glu Ala Leu Ala Ala Gly Gly Ser Cys

20 25 30

(2) INFORMATION FOR SEQ ID NO: 9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 34 amino acids

(B) TYPE: amino acid

(C) STANDEDNESS: single

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptides

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

Gly Leu Phe Gly Ala Ile Ala Gluy Phe Ile Glu Asn Glu Trp Glu Gly

1 5 10 15

Leu Ala Glu Ala Leu Ala Glu Ala Leu Glu Ala Leu Ala Ala Gly Gly Ser Cys

20 25 30

(2) INFORMATION FOR SEQ ID NO: 10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 34 amino acids

(B) TYPE: amino acid

(C) STANDEDNESS: single

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptides

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

Gly Leu Phe Glu Ala Ile Glu Gly Phe Ile Glu Asn Gly Trp Glu Gly

1 5 10 15

Leu Ala Glu Ala Leu Ala Glu Ala Leu Glu Ala Leu Ala Ala Gly Gly Ser Cys

20 25 30

(2) INFORMATION FOR SEQ ID NO: 11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 amino acids

(B) TYPE: amino acid

(C) STANDEDNESS: single

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptides

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:

Gly Leu Phe Glu Ala Ile Glu Gly Phe Ile Glu Asn Gly Trp Glu Gly

1 5 10 15

Met Ile Asp Gly Gly Gly Cys

20

(2) INFORMATION FOR SEQ ID NO: 12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 36 amino acids

(B) TYPE: amino acid

(C) STANDEDNESS: single

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptides

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:

Gly Ile Gly Ala Val Leu Lys Val Leu Thr Thr Gly Leu Pro Ala Leu

1 5 10 15

Ile Ser Trp Ile Lys Arg Lys Arg Gln Gln Lys Lys Lys Lys Lys Lys Lys Lys Lys

20 25 30 35

(2) INFORMATION FOR SEQ ID NO: 13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 36 amino acids

(B) TYPE: amino acid

(C) STANDEDNESS: single

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptides

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:

Gly Ile Gly Ala Val Leu Glu Val Leu Glu Thr Gly Leu Pro Ala Leu

1 5 10 15

Ile Ser Trp Ile Lys Arg Lys Arg Gln Gln Lys Lys Lys Lys Lys Lys Lys Lys Lys

20 25 30 35

Claims (95)

1. A composition for transfection of higher eukaryotic cells with a complex of nucleic acid and a substance having an affinity for nucleic acid, which substance can be associated with an internalizing factor for said cells, characterized in that said composition contains an agent that has the ability to be externalized into cells, either "per se" or as a component of the nucleic acid complex, and to release the contents of the endosome, in which the complex is located after entering the cell, into the cytoplasm. 2. The composition according to claim 1, characterized in that the endosomolytic agent is a virus or a viral component that is an internalizing factor "per se" for said cells. 3. The composition according to claim 1, characterized in that endosomolytic agents bind nucleic acid to a domain or is associated with a substance that has an affinity for nucleic acid and has the ability to be internalized into said cells as a component of the conjugate/nucleic acid complex, and said complex further may contain internalizing factor for cells. 4. Composition according to claim 3, characterized in that the endosomolytic agent is covalently bound to a substance that has an affinity for nucleic acid. 5. Composition according to claim 3, characterized in that the endosomolytic agent is not covalently linked to a substance having an affinity for nucleic acid. 6. Composition according to claim 5, characterized in that the connection is made via a biotin-streptavidin bridge. 7. Composition according to claim 5, characterized in that the coupling is achieved by an ionic method. 8. The composition according to claim 3, characterized in that the endosomolytic agent is bound to the nucleic acid directly via the linking domain of the nucleic acid. 9. Composition according to claim 3, characterized in that the endosomolytic agent is a virus or a viral component. 10. Composition according to claims 2 and 9, characterized in that the endosomolytic agent is an adenovirus. 11. The composition according to claim 10, characterized in that the adenovirus is a mutant. 12. The composition according to claim 11, characterized in that the adenovirus is an irresponsible mutant for recovery. 13. The composition according to claim 12, characterized in that the adenovirus has one or more mutations and/or breaks in the ElA region. 14. Composition according to claim 10, characterized in that the adenovirus is inactivated. 15. Composition according to claim 14, characterized in that the adenovirus is inactivated by short-wave UV radiation. 16. Composition according to claim 14, characterized in that the adenovirus is inactivated by UV/psoralen. 17. Composition according to claim 14, characterized in that the adenovirus is inactivated by formaldehyde. 18. Composition according to claim 9, characterized in that the viral component is one or more adenovirus proteins. 19. Composition according to claim 2 or 9, characterized in that the virus is a picorna virus, primarily a rhinovirus. 20. Composition according to claim 19, characterized in that the rhinovirus is inactivated. 21. Composition according to claim 3, characterized in that the endosomolytic agent is not an internalizing factor "per se" for the cell and that the nucleic acid complex additionally comprises an internalizing factor for the cell in question, which factor is bound to a substance that has an affinity for nucleic acid. 22. The composition according to claim 21, characterized in that the endosomolytic agent is a virus or a viral component that is not an internalizing factor for a human cell. 23. The composition according to claim 22, characterized in that the endosomolytic agent is a virus that infects non-human species. 24. Composition according to claim 23, characterized in that the virus is an adenovirus. 25. The composition according to claim 24, characterized in that the adenovirus is avian. 26. The composition according to claim 23, characterized in that the adenovirus is a virus of a postmortem chicken embryo. 27. Composition according to claim 21, characterized in that the endosomolytic agent can be a modified endosomolytic viral peptide. 28. Composition according to claim 27, characterized in that the endosomolytic peptide is the hemagglutinin HA2 influenza peptide. 29. The composition according to claim 28, characterized in that the peptide has the sequence Gly Leu Phe Glu Ala Ile Ala Gly The Ile Glu Asn Gly Trp Glu Gly Met Ile Asp Gly Gly Gly Cys. 30. Composition according to claim 28, characterized in that the peptide has the sequence Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Asn Gly Trp Glu Gly Met Ile Ap Gly Gly Gly Cis. 31. Composition according to claim 21, characterized in that the endosomolytic agent is a non-viral, optionally modified natural or synthetic peptide. 32. Composition according to claim 27 or 31, characterized in that the peptide has a nucleic xylin binding domain. 33. Composition according to claim 3 or 21, characterized in that the internalizing factor is transferrin. 34. Composition according to claim 3 or 21, characterized in that the internalizing factor is a ligand for hepatocytes. 35. The composition according to claim 34, characterized in that the internalizing factor is a ligand for the asialoglycoprotein receptor. 36. Composition according to claim 35, characterized in that the internalizing factor is tetra-galactose-polylysine. 37. Nucleic xylin complex usable as a constituent of the composition according to all claims 3 to 36, with the said complex including one or more nucleic xylins to be expressed in the cell, an endosomolytic agent that originally has a nucleic acid binding domain or that is attached to a substance having an affinity for nucleic acid, which complex may additionally contain an internalizing factor linked to a substance having an affinity for nucleic acid. 38. Complex according to claim 37, characterized in that the nucleic acid is therapeutically active. 39. Complex according to claim 38, characterized in that said nucleic acid comprises one or more DNA molecules that are active in gene therapy. 40. Complex according to claim 39, characterized in that said DNA molecules encode cytokines. 41. Complex according to claim 38, characterized in that said nucleic acid contains a series of nucleotides, from which RNA molecules can be translated, which specifically inhibit cellular functions. 42. Complex according to claim 37, characterized in that the substance, which has an affinity for nucleic xylin, is an organic polycation. 43. Complex according to claim 42, characterized in that it is a polycation, polylysine. 44. The complex according to claim 37, characterized in that both the endosomolytic agent and the internalizing factor are linked to the same substance, which has an affinity for nucleic acid. 45. Complex according to claim 44, characterized in that both the endosomolytic agent and the internalizing factor are bound to polylysine. 46. Complex according to claim 45, characterized in that it additionally contains non-covalently bound polylysine. 47. Conjugate usable as a constituent of the complex according to claims 37 to 46, characterized in that said conjugate comprises an endosomolytic agent which is bound to a substance having an affinity for nucleic acid. 48. Conjugate according to claim 47, characterized in that the endosomolytic agent is covalently bound to a substance having an affinity for nucleic acid. 49. The conjugate according to claim 47, characterized in that the endosomolytic agent is non-covalently bound to a substance having an affinity for nucleic xylin. 50. Conjugate according to claim 49, characterized in that the connection is performed via a biotin-strptavidin bridge. 51. Conjugate according to claim 49, characterized in that the connection is ionic. 52. Conjugate according to claim 47, characterized in that the endosomolytic agent is a virus or a viral component. 53. Conjugate according to claim 52, characterized in that the endosomolytic agent is an adenovirus. 54. The conjugate according to claim 53, characterized in that the adenovirus is a mutant. 55. Conjugate according to claim 54, characterized in that the adenovirus is an irresponsible mutant for recovery. 56. Conjugate according to claim 55, characterized in that the adenovirus has one or more mutations and/or breaks in the E1A region. 57. Conjugate according to claim 53, characterized in that the adenovirus is inactivated. 58. Conjugate according to claim 57, characterized in that the adenovirus is inactivated by short-wave UV radiation. 59. Conjugate according to claim 57, characterized in that the adenovirus is inactivated by UV/psoralen. 60. Conjugate according to claim 57, characterized in that the adenovirus is inactivated by formaldehyde. 61. Conjugate according to claim 52, characterized in that the viral component is an adenoviral protein. 62. Conjugate according to claim 52, characterized in that the virus is a picorna virus, especially a rhinovirus. 63. Conjugate according to claim 62, characterized in that the rhinovirus is inactivated. 64. The conjugate according to claim 47, characterized in that the endosomolytic adenovirus is not an internalizing factor per se, for the cell to be transfected. 65. The conjugate according to claim 64, characterized in that the endosomolytic agent is a virus or a viral component that is not an internalizing factor for a human cell. 66. The conjugate according to claim 64, characterized in that the endosomolytic agent is a virus that is infectious for non-human species. 67. Conjugate according to claim 66, characterized in that the virus is an adenovirus. 68. Conjugate according to claim 67, characterized in that the adenovirus is avian. 69. Conjugate according to claim 68, characterized in that the adenovirus is a post-mortem chicken embryo virus. 70. Conjugate according to claim 64, characterized in that the endosomolytic agent can be a modified endosomolytic viral peptide. 71. Conjugate according to claim 70, characterized in that the endosomolytic agent is the influenza HA2 hemagglutinin peptide. 72. Conjugate according to claim 71, characterized in that the peptide has the sequence Gly Leu Gly Ala Ile Ala Gly Phe Ile Glu Asn Gly Trp Glu Gly Met Ile Asp Gly Gly Gly Cys. 73. Conjugate according to claim 71, characterized in that the peptide has the sequence Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Asn Gly Trp Glu Gly Net Ile Asp Gly Gly Gly Cys 74. The conjugate according to claim 47, characterized in that the endosomolytic agent is a non-viral, optionally modified natural or synthetic peptide. 75. An endosomolytic peptide usable as an ingredient of the composition of claim 32, characterized in that it has an endosomolytic domain and a nucleic acid binding domain. 76. The endosomolytic peptide of claim 75, characterized in that the nucleic acid binding domain is an oligolysine extension. 77. The method for preparing the conjugate from claim 48, characterized in that the virus or (poly)peptide endosomolytic agent and the polyamine are enzymatically linked in the presence of transflutaminase. 78. The method for preparing the conjugate from claim 48, characterized in that the virus or (poly)peptide endosomolytic agent and the polyamine are chemically linked. 79. The process for preparing the conjugate from claim 49, which includes a) modification of virus or viral component with biotin i b) connecting the modified virus or the modified viral component obtained in phase a), to the treptavidin-bound polyamine. 80. A method for introducing nucleic acid into eukaryotic cells, characterized in that the cells are related to the composition according to any of claims 1 to 36. 81. The method according to claim 80, characterized in that the cells are human cells. 82. The method according to claim 81, characterized in that the cells are human tumor cells. 83. The method according to claim 81, characterized in that the cells are human myoblasts. 84. The method according to claim 81, characterized in that the cells are human fibroblasts. 85. The method according to claim 81, characterized in that the cells are human hepatocytes. 86. The method according to claim 81, characterized in that the cells are human endothelial cells. 87. The method according to claim 81, characterized in that the cells are human lung epithelial cells. 88. The method according to claim 81, characterized in that the nucleic acid of the DNA composition is active in gene therapy. 89. The method according to claims 82 and 88, characterized in that the tumor cells are in contact with the composition "ex vivo" and that the mentioned DNA encodes one or more immunomodulating substances, especially cytokines. 90. A method of preparing a protein of interest in a higher eukaryotic cell, characterized in that the cells are treated with the composition of claim 1, that the nucleic acid comprises a DNA sequence encoding the desired protein, that the cells are grown under conditions suitable for protein expression, and that the protein regenerates. 91. A pharmaceutical composition comprising the composition of any one of claims 1 to 36, wherein the nucleic acid is a therapeutically active and pharmaceutically acceptable carrier. 92. Transfection kit, characterized in that it contains the constituents of the nucleic acid complex of the composition from claim 1 and/or the endosomolytic agent of the said composition or the constituents of the said endosomolytic agent, as separate or partially separated components. 93. The transfection kit from claim 92, characterized in that it contains the adenovirus-polylysine conjugate bound by transglutaminase, as a separate constituent. 94. The transfection kit of claim 92, characterized in that it contains a biotin-modified endosomolytic agent and a steptavidin-modified substance having an affinity for nucleic xylin as a separate constituent for the conjugates immediately before administration. 95. The transfection kit of claim 94, characterized in that it contains biotinylated adenovirus and streptavidin-polylysine.