DE4418825A1 - Transfection of eukaryotic cells - Google Patents

Abstract

Process for introducing foreign material, e.g. nucleic acids, into higher eukaryotic cells comprises introducing the foreign material and transfection components into the cells and: (a) introducing \- 1 nucleic acid (esp. DNA) mol. whose expression prods. at least partially block apoptosis induced by introduction of the foreign material and/or (b) at least partially inhibiting the inflammatory response to the cells by (i) treating the cells externally with \- 1 antiinflammatory substance and/or (ii) introducing \- 1 antiinflammatory substance, or nucleic acid (esp. DNA) mol., coding for such substances, into the cells. Also claimed is a transfection complex comprising: (1) a nucleic acid mol. to be expressed in a target cell, where the nucleic acid is complexed with a polycation and the polycation is opt. conjugated with a ligand for the target cell; (2) an adenovirus or an adenovirus-polycation conjugate, and (3) a DNA mol. with anti-apoptotic activity and/or a DNA mol. coding for a substance with antiinflammatory activity.

Description

The present invention relates to a Method for introducing foreign material, in particular nucleic acid, in higher eukaryotic Cells.

Need for an efficient system for insertion of nucleic acid in living cells exists mainly in Frame of gene therapy. This involves genes in cells introduced in vivo the synthesis therapeutically to achieve effective gene products.

Standard methods for the transfection of cells use u. a. Calcium phosphate (for applications in vitro), cationic lipids (Felgner et al., 1993) or Liposomes.

The hitherto most advanced technologies in the context of gene therapy use recombinant Vector systems (retroviruses or adenoviruses) for the Transfer of genes into the cell (Wilson et al., 1990; Kasid et al., 1990, WO 93/03769).

Alternative strategies for gene transfer lie Mechanisms underlying the cell for the Transport of macromolecules served. An example this is the import of genes into the cell via the Pathway of receptor-mediated endocytosis (eg, Wu and Wu, 1987, Wagner et al., 1990, and EP-A1 0388 758).

Overviews of gene therapy in general and about non-viral gene transfer methods in particular are by Mulligan, 1993, and by Cotten and Wagner, 1993, given.

For gene transfer with DNA / polycation complexes By receptor-mediated endocytosis was a  Improvement suggested the use of Components due to their ability provides the Be able to release content of endosomes, z. B. Adenoviruses or fusogenic peptides. The use of endosomolytic components causes an increase the efficiency of gene transfer, by reducing the in the cell internalized DNA complexes in the Lysosomes is avoided (Curiel et al., 1991, Curiel et al., 1992a; Zatloukal et al., 1992; Cotten et al. 1992; Wagner et al., 1992; Curiel et al., 1992b; WHERE 93/07283). Et al it was suggested the adenoviruses by binding to polylysine. The Adenovirus-polylysine conjugates can be used together with Transferrin polylysine conjugates with DNA complexed with ternary transferrin Polylysine / adenovirus polylysine / DNA complexes are formed (Wagner et al., 1992). The complexes bind Transferrin and adenovirus receptors on the Target cells. After endocytosis causes the adenovirus the departure of endosomes, that has the release of the Material from the endosome into the cytoplasm result. These Technique, also referred to as "transferrinfection" is, as opposed to conventional viral techniques increased safety (Cotten et al., 1992).

With adenovirus-assisted gene transfer at the Basis of receptor-mediated endocytosis were Although transiently high expression levels achieved, it is but so far due to toxic effects in some Cell types failed to express over one to obtain a longer period of time. These toxic effects are due to various defensive responses of the host cell, soon after the virus enters the cell, due. The mechanisms that activate these answers are not yet cleared up, Investigations with replication-deficient  Adenovirus mutants suggest that the answers of the host take place very early, possibly even before virus gene expression.

One component of this host response is one Activation of "interferon responsive genes" (genes, that respond to interferon), most likely by activation of ISGF3, one on interferon responsive transcription factor whose Binding sites are upstream of a series of Interferon responsive genes are located. One of the activated genes is the protein kinase p68, which is activated by double-stranded RNA. p68 will be in synthesized in an inactive form and is subject in Presence of dsRNA in an autocatalytic Phosphorylation, which activates the kinase, leading to Phosphorylation and inactivation of the translation Initiation factor eIF2a performs, thereby initiating the protein translation is blocked. It was also reports that NF-κB is activated by dsRNA (Visvanathan and Goodbourn, 1989) suggests that p68 may phosphorylate directly IKB and NF-κB activated.

The type C adenoviruses have two strong mechanisms to prevent this translation arrest by the host:
The Ela gene products can directly interfere with the activation of ISGF3 (Gutch and Reich, 1991, Ackrill et al., 1991).

A second, more direct-acting mechanism uses the VA1 genes. The gene product forms a stable secondary structure attached to the p68 kinase can bind without activating the kinase, and which  the binding and activation by the actual Activators can prevent (Manche et al., 1992; Mathews and Shenk, 1991).

Another inflammatory reaction mechanism as Consequence of the onset of virus is likely in activation of the inflammatory transcription factor NF-IL-6 and the secretion of the inflammatory cytokine IL-6 (Sehgal et al., 1988; Kishimoto et al., 1992). This factor in turn activates, often in interaction with NF-κB, the IL-6 gene itself as well as a number other inflammatory response genes such as TNF and IL-1. Recently it was reported that IL-6 in turn the interferon-responsive transcription factor IRF 1 ("Interferon Response Factor 1") (Harroch et al., 1994). This may be for either Interferon response to the entry of adenovirus be responsible or strengthen it. Because the most of the early genes of adenovirus type C. One could include binding sites for NF-IL-6 assume that the activation of NF-IL-6 by Adenovirus triggering the virus gene Guaranteed expression cascade.

Another line of defense of the host cell is likely be the apoptotic answer. It has long been known that mutations mapping in the E1B 19K region for the deg phenotype, an enhanced cytopathic Effect of decomposing the chromosomal DNA of the host accompanies (D'Halluin et al., 1979, Ezoe et al. 1981; Lai Fatt and Mak, 1982; Pilder et al., 1984; Subramanian et al., 1984; Takemori et al., 1984; White et al., 1984). junior Studies have identified apoptosis, which by expression of an E1A growth signal in Absence of E1B is evoked (White and  Stillman, 1987; White et al., 1994; Rao et al., 1992); at this apoptosis is likely triggered by E1A Release of the transcription factor E2F from the Rb Protein be involved (Wu and Levine, 1994). It was demonstrated that the E1B 19K protein is highly potent Analogous to the apoptosis-blocking host gene Bcl-2 (Rao et al., 1992, Debbas and White, 1993). The expression of each of these proteins can be a triggered by various signals apoptosis To block. In connection with the investigations with Myc was found to be a cell of growth signals either for proliferation, if appropriate Amplification signals, e.g. As Ras or Src available stand, or for apoptosis, when the Myc signals are present alone (Evan et al., 1992), determine. A similar model for through Adenovirus E1-induced transformation is due Experiments supported that show that apoptosis by E1A expression triggered in the absence of E1B 19K (White and Stillman, 1987; Rao et al., 1992; Debbas and White, 1993). A recent one Investigation of Sindbis virus infection has shown that the expression of Bcl-2 in the host cell decisive factor for the outcome of a Viral infection (Levine et al., 1993): When Absence of Bcl-2, the cell undergoes apoptosis, bringing the virus production to a short acute phase is limited. In the presence of Bcl-2 expression There is a chronic virus production, because the apoptotic response to the acute phase infection absent. Anti-apoptotic activities were further explored in Epstein-Barr virus (the BHER1 gene, Pearson et al. 1987), baculovirus (the p35 gene, Sugimoto et al. 1994) and in the African swine fever virus (the LMW5-HL gene; Neilan et al., 1993). This suggests that the apoptotic response to the  Virus infection occurs in many cases and viruses Strategies have developed to this answer too To block.

The inactivated adenovirus capsids for the Gene transfer by receptor-mediated endocytosis The experiments carried out were initially based on the Assume that the viral capsid is only as one of the endosomes disrupting reagent acts as a function of Surface proteins of the virus capsid is. There were Inactivation methods developed that the Block virus transcription and replication, without to alter the endosomolytic activity of the capsid (Cotten et al., 1994). In the course of further experiments with Adenovirus-assisted transfections were the Contamination with lipopolysaccharide (LPS) as one Source of toxicity in primary cell transfections identified. This toxicity problem could be solved by careful removal of LPS from the DNA easily be remedied. Although the inactivation of the virus and the removal of LPS two forms of viral eliminated toxicity all cell types long-term gene expression, not even with Psoralen / UV-inactivated human adenovirus in Presence of LPS-free DNA.

The present invention was based on the object To elucidate the mechanisms which at Transfections observed toxicity effects occur on the basis of the gained Findings a new, improved method for the Introducing foreign material, in particular Nucleic acids in the higher eukaryotic cell provide.  

Previous efforts to improve the Gene transfer methods focused on the Amplification of gene expression. So z. B. reports, that VA1 expression is the gene expression of co transfected genes by using the Stability mRNA or its amount increased (Svensson and Akusjärvi, 1984; Støker et al., 1989; Svensson and Akusjärvi, 1990; Mathews and Shenk, 1991). The one for it Responsible mechanism is at the molecular level not yet cleared up; a connection with Toxicity problems should not be given.

The present invention relates to a method for Introducing foreign material, in particular Nucleic acid, in higher eukaryotic cells, where one into the cell except the foreign material and the transfection components

• a) one or more nucleic acid molecules, In particular, introduces DNA molecules whose Expression products by introducing the Foreign material induced apoptosis of the host cell at least partially block, and / or that one
• b) the inflammatory response of the cells at least partially inhibited by
• i) the cells externally with one or more anti treated inflammatory substance (s) and or
• ii) one or more anti inflammatory substance (s) or for that encoding nucleic acid, especially DNA molecules brings.

The DNA molecules defined in a) are described below as "anti-apoptosis genes" or as "DNA molecules with anti-apoptosis effect ".  

In a preferred embodiment, the inventive method to the above, from Curiel et al., 1991; Curiel et al., 1992a; Zatloukal et al., 1992; Cotten et al., 1992; Wagner et al., 1992; Curiel et al., 1992b and in WO 93/07283 described method applied to the DNA using of conjugates of a ligand for the target cell and a polycation with the participation of, optionally conjugated with a polycation, adenovirus as endosomolytic agent is introduced into the cell. These publications are expressly incorporated into the Disclosure of the present invention included. (This method is referred to below as "adenovirus supported transferrin infection ").

The inventive method, however, can all methods of introducing foreign material into the cell where those set out above Toxicity effects occur, be applied, in particular to gene transfer methods, e.g. B. in the Use of recombinant adenovirus vectors. such Gene transfer methods are used in vitro as well as in gene therapy both in vivo and ex vivo, especially in fibroblasts, hematopoietic cells, Endothelial cells or pulmonary epithelial cells.

In a preferred embodiment of the invention that is anti-apoptosis gene, the human Bcl-2 gene (Seto et al., 1988).

In a further preferred embodiment that is anti-apoptosis gene the E1B 19K gene of adenovirus type 2 or 5 (White and Cipriani, 1990).

Further examples in the context of the present Invention suitable anti-apoptosis genes are the BHRF1  Gen of Epstein-Barr virus (Pearson et al., 1987) baculovirus p35 gene (Sugimoto et al., 1994) LMW5 HL gene from African swine fever virus (Neilan et al., 1993) and the ced-9 gene of Caenorhabditis elegans (Sugimoto et al., 1994).

These gene products act in the apoptosis Signal transmission path relatively far downstream, d. H. close to those at the degradation processes immediately involved proteases. So z. B. Bcl-2 and Bcl-2 Similar proteins are believed to activate the proteases designated as ICE or ICE-like To block. Since this protease (s) apparently a whole crucial function for apoptosis comes also a crucial role in their blocking.

Except the substances with a Bcl-2-like anti- Apoptosis mechanism can also be considered those that a component located further upstream in the apoptosis Signal transmission path acts block. Such Component is z. B. p53, whose activation is one of Signal transmission pathways triggers apoptosis to lead. If so by the transfection process triggered apoptosis via activation of p53, could substances that inactivate p53 for the Blocking apoptosis can be used, wherein one thing to note is that the blocking is not through another signal transmission, also on the other hand, that leads to apoptosis, is bypassed the inactivation of p53 in the particular cell type does not cause a transformed state.

Examples of genes that inactivate p53 are E1B 55K of adenovirus type 2 or type 5 (White and Cipriani, 1989) or E1 functions of other adenovirus strains, z. Adenovirus 12 or the CELO virus (Chick embryo  Lethal Orphan Virus), the human E6 gene Papillomavirus-16 (Levine, 1990), the large T-antigen from SV 40 (McCarthy et al., 1994), the mdm-2 gene (Momand et al., 1992; Oliner et al., 1993; Chen et al., 1994).

In the preferred application of the invention Procedure on the adenovirus-assisted Transferinfection becomes the anti-apoptosis gene simple way in the form of a plasmid together with the Plasmid containing the DNA sequence, their expression in the cell to achieve the desired effect (im hereinafter referred to as "effective DNA"), e.g. B. a therapeutically effective gene into which Transfection complexes installed. It is then together with the plasmid carrying the effective DNA, complexed, z. B. with transferrin-polylysine and Adenovirus polylysine, before. The molar ratio of effective gene and anti-apoptosis gene can by means of Titration can be determined by otherwise identical conditions over a longer period of time the expression of the active gene at different Proportions of anti-apoptosis gene is measured. As part of The present invention has a ratio of 5: 1 chosen that has proved favorable. Because the Gene products of some anti-apoptosis genes very strong Show effect, even smaller proportions can be used become. A due to a possible toxicity Upper limit can also be determined by titration become.

It is also possible to use the effective DNA sequence and the anti-apoptosis gene together in a single plasmid too integrate, in this case, the molar Ratio of the two genes 1: 1, if in each case a copy on the plasmid.  

In the context of the present invention was found that cell death due to the entry of Adenovirus in the absence of viral gene expression is triggered. The simultaneous introduction of a active adenovirus E1 region provided for a Relief from apoptosis; Experiments with plasmids, carrying the single E1 genes, identified E1B 19K as the gene responsible for the protective effect. Furthermore, the apoptosis phenotype could be shown that the simultaneous introduction of the Bcl-2 gene can also block the toxicity.

To activate the inflammatory response to study the entry of adenovirus were in Within the scope of the present invention, two cell lines made the luciferase gene under the control from promoters that are based on two of the most important ones activated during the inflammation Respond to transcription factors, namely NF-IL-6 and NF-kB. Thus, the condition was created, the Activation of these transcription factors as a response on the entry of adenovirus or the Transfection complex to investigate quickly. To the Activation of NF-κB was a construct used that the luciferase gene under the control a simple TATA box and three NF-κB Contains binding elements. Furthermore, a study on NF-IL 6 / NF-κB responsive, driven by the IL-6 promoter Luciferase construct used. The Activation behavior of the two reporter cell lines confirmed that the promoters except on their already earlier activators also characterized LPS and PMA on adenovirus, both non-inactivated also psoralen / UV-inactivated (Table 1). It turned out that activation by psoralen  inactivated virus to the same extent as with non- inactivated virus. This supports the theory that it is not the early virus gene expression that activated these factors, but rather the binding and the entry of adenovirus.

For the measure defined in step b) under i) external treatment with anti-inflammatory Substances comes z. As the treatment with retinoic acid ("Retinoic acid") or other retinoids, in consideration, which was reported to induce IL-1 Inhibit production of IL-6 by lung fibroblasts (Gross et al., 1993; Zitnik et al., 1994). The Effect of this class of substances is likely insist that they increase the concentration of IL-6 mRNA control.

Another class of substances for the inhibition of inflammatory host response are the Glucocorticoids which have been shown to be the Production of IL-6 and / or IL-8 in response to inhibit inflammatory stimuli (Ray et al., 1990; Barber et al., 1993; Wertheim et al., 1993).

In the context of the present invention has been shown be that the glucocorticoid dexamethasone the Gene expression both alone and in interaction reinforced with the anti-apoptosis E1B 19K gene.

The treatment of the cells can also be done with anti inflammatory polypeptides, such as IL-10 or TGF-β take place.

For the external treatment of the cells, the anti inflammatory substance appropriate to the medium added.  

According to step b) ii) the anti-inflammatory Substance as such or in a preferred Embodiment in the form of DNA coding for it introduced the cell, z. In the form of a plasmid, the one for an anti-inflammatory protein like IL-10 (Moore et al., 1990) or TGF-β (Massaque et al., 1987). contains coding sequence.

One preferred in the context of the present invention used anti-inflammatory substance is VA1. Adenovirus VA1 RNA is a small molecule of RNA Polymerase III synthesized RNA molecule used for the efficient expression of the virus genome responsible for.

In light of reports that double-stranded RNA is NF-κB activated (Visvanathan and Goodbourn, 1989) and the known effect VA1 RNA, the activation of dsRNA to block activated kinase p68 was investigated, whether the introduction of a VA1 gene is the activation of NF-κB caused by the entry of Transfection complexes from adenovirus / polylysine / DNA is triggered, blocked.

It was within the scope of the present invention found that the expression of the adenovirus VA1 Gene (caused by incorporation of VA1 encoding DNA into the transfection complex) Activation of NF-κB by adenovirus 1.5 times of the base value decreased. It can thus the Expression of the adenovirus VA1 gene during the Transfection can be used to activate NF-κB, which is triggered by the transfection process is going to modulate.  

The made in the context of the present invention Observation that VA1 expression is the activation inhibits inflammatory cytokines, is likely to be one of increasing the stability of the mRNA or its amount, as reported for the calcium phosphate method, independent phenomenon. At this Phenomenon is possibly the inhibition of p68 Kinase activation decisively involved.

The VA1 can act as an RNA molecule or, in a preferred Embodiment, in the form of the VA1 DNA in the cell Imported. In this case, a plasmid containing the VA1 Sequence, optionally in multiple copies, contains together with the plasmid with the effective DNA and the Plasmid with the anti-apoptosis gene in Transfection complexes are integrated. It is also possible, two of the three sequences, or even all three, on a single plasmid.

An example of one in the context of gene therapy applicable triple combination would be a combination of the factor VIII sequence, an anti-apoptosis gene like bcl-2 or E1B 19K and VA1 DNA.

As an alternative to VA1 DNA, genes can be used have a VA1 corresponding effect, z. B. EBER's Epstein Barr Virus (Clarke et al., 1990; Clarke et al. 1991) or the TAR RNA from the HIV virus (Gunnery et al., 1990).

LIST OF FIGURES

Fig. 1 Time course of the activation of NF-IL-6 and NF-κB,

FIG. 2 blocking the activation of NF-κB by expression of VA1, FIG.

FIG. 3 blocking the activation of the IL-6 promoter by expression of VA1, FIG.

Figure 4 Influence of expression of E1A, E1A 13S, E1B, E1B 19K and Bcl-2 on gene expression.

Fig. 5 Effect of the expression of E1B 19K, E1A and Bcl-2 on gene expression at different virus doses,

Fig. 6 Effect of the expression of Bcl-2, E1B 19K, E1A and E1A 13S on the long term gene expression,

Fig. 7 Effect of transfection on the morphology of fibroblasts,

Figure 8 Inhibition of adenovirus or LPS-induced toxicity by pre-expression of E1B 19K and Bcl-2.

In the following examples were, if not otherwise indicated the following materials and methods used:

a) DNA plasmids i) expression plasmid for the adenovirus VA1 sequence

Initially, the entire E1 region was adenovirus type 5 (nucleotide 1-5778) obtained by the adenovirus d11014 DNA (Bridge and Ketner, 1989) with XhoI plus Ase1 was digested. This fragment was mixed with Klenow Treated polymerase and in the SmaI site of the Plasmid pAALM (obtained from pSP64 (Boehringer Mannheim) by its small EcoRI / PvuII fragment (bp 53 - bp 232) has been replaced by EcoRI / PvuII- Fragment (bp59-bp104) of pSPT18 (Boehringer  Mannheim)) to obtain pE1paalm. The VA1 expression plasmid pXVAH was constructed the 5324 bp HindIII fragment (nucleotide 6241- 11656) of the adenovirus DNA into the HindIII site of pAALM was inserted to obtain pHVAH. pHVAH was then split with XBaI and religated to To get pXVAH (this became a big piece, from the XbaI site in pAALM to the XbaI site Position 10589 in the adenovirus sequence). The clone pXVAH contained the adenovirus sequence of Nucleotide 10589-11565.

ii) luciferase reporter plasmid pNF-κB-Luc

The NF-κB-responsive plasmid pNF-κB-Luc was described as a derivative of Boehmelt et al., 1992, constructed plasmid pTK3kbB. This was pTK3kbB cut with XhoI and NcoI to the most to remove the CAT coding sequence with Klenow enzyme treated and ligated with a Klenow treated HindIII / SspI fragment of pRSVL containing the luciferase sequence (De Wet et al., 1987). This resulted the plasmid p3K-Luc, which has a Triple binding site for the transcription factor NF- κB plus the luciferase sequence, driven by a Thymidine kinase (TK) TATA box containing.

iii) Luciferase construct pIL-6-Luc

It has been described by Matsusaka et al., 1993 Construct that is driven by the IL-6 promoter Contains luciferase sequence.

iv) Expression plasmid for E1A 13S

It has been described by Kedinger et al., 1984 Expression plasmid of the name p13S, which contains the endogenous E1A promoter used to express the E1A 13S cDNA to drive used.

v) Expression plasmids for E1A, E1B and E1B 19K

The expression plasmids used the term pCMVE1A, pCMVE1B and pCMV19K were from White and Cipriani, 1989 and 1990.

vi) expression plasmid for Bcl-2

The expression plasmid designated pCMV-Bcl-2 was prepared by flanking the human Bcl-2 cDNA flanked by two EcoRI sites (Seto et al., 1988), downstream from a CMV immediate early promoter in the Expression plasmid pBK-CMV (Stratagene) was cloned.

vii) plasmid DNA pCMVL

The construction of the plasmid is described in WO 93/07283 described.

viii) plasmid DNA pCMVβgal

It became that described by Lim and Chae, 1989 Plasmid used.

b) Adenovirus d11014 DNA preparation

The E4-deficient adenovirus 5, d11014 (Bridge and Ketner, 1989) was in the complementing cell line W162 (Weinberg and Ketner, 1983). Pellets of infected cells were added to 2 ml / 2x10⁷ cells in  20mM HEPES, pH 7.4, 1mM PMSF (Phenylmethylsulfonyl fluoride) and three Freeze-thaw cycles (liquid nitrogen, 37 ° C) subjected. The suspension was then washed with a equal volume of freon, vortexed and 10 min long at 3,000 rpm (Heraeus Sepatech, 2705 rotor) centrifuged. The aqueous (upper) phase became lifted, and the freon phase was 1/5 volume 20 mM HEPES, pH 7.4 vortexed and again centrifuged. The aqueous phases were combined, in a Beckman VTi50 centrifuge tube (15 ml / tube) transferred and with 15 ml of 1.2 g / cm³ CsCl / 20 mM HEPES, pH 7.4 and 7 ml 1.45 g / cm³ CsCl, 20 mM HEPES pH 7.4 underlaid. The samples were kept at 20 ° C for 40 min in a Beckman VTi5O rotor at 49,000 rpm centrifuged. The lower opalescent band of mature virus particles at 1.34 to 1.35 g / cm³ (measured by refractive index) and the upper band (immature Particles at 1.31 to 1.32 g / cc) were separated collected and an equilibrium centrifugation (more as 4 h) at 63,000 rpm in a VTi65 rotor subjected. The opalescent virus bands (either 1.31 g / cc immature or 1.34 g / cc mature) harvested. The biotinylation of the obtained Virus particles with N-hydroxysuccinimide-biotin (Pierce), inactivation with 8-methoxypsoralen / UVA and the Purification by gel filtration using a Pharmacia PD10 column, equilibrated with HBS / 40% Glycerol were performed as in WO 93/07283 Wagner et al., 1992, and Cotten et al., 1992, described. The virus samples were quantitatively transferred over the protein concentration is determined (Biorad Bradford Assay with BSA as standard), with the Relationship 1 mg / ml protein = 3.4 × 10¹² Adenovirus particles / ml (Lemay et al., 1980) has been.  

c) Transferrin-polylysine conjugates (TfpL)

For the synthesis of conjugates of transferrin and Polylysine with a chain length of 290 lysine residues became the method described by Wagner et al., 1991 used.

d) streptavidin-polylysine conjugates (StpL)

The conjugates were prepared as in WO 93/07283 using polylysine 250 has been.

e) adenovirus / DNA complexes

Samples of biotinylated, psoralen / UV-inactivated Adenovirus d11014 (8 μl, 1 x 10 12 particles / ml) diluted in 150 μl HBS and with 1 μg StpL in 150 μg HBS Mixed at room temperature for 30 minutes. Aliquots of 6 μg Plasmid DNA (in the case of mixtures of pCMVL DNA and another plasmid was 5: 1) diluted in 100 ul HBS and then with the Adenovirus / StpL solution for 30 min at room temperature mixed. Finally, a 100 μl aliquot of HBS containing 5 μg TfpL, added to each sample, followed by incubation at room temperature for 30 minutes. Aliquots of these transfection complexes were then, as individually described in the respective examples the cells abandoned (generally 10 to 30 μl / 20,000 cells).

f) reporter cell lines

For the preparation of the clonal cell lines containing the reporter plasmid pNF-κB-Luc or pIL-6-Luc  of the human lung carcinoma cell line A549 (ATCC CCL 185). The plasmids were incubated with the neomycin Phosphotransferase expression plasmid of the term pMuatt (a pUC19 plasmid containing the TKneo) Sequence; Grosveld et al., 1982) using the Transfectam method (Behr et al., 1989) co-transfected and on cells resistant to 400 μg / ml G418 (Sigma) are selected. Clonal derivatives, the Luciferase expressed after induction by PMA, were identified and expanded.

In a similar manner, A549 cells were obtained which had the Expression plasmid for Bcl-2 or E1B 19K included, with the difference that that Neomycin phosphotransferase plasmid pSVneo (Clontech) instead of pMuatt and that instead of pure populations pools of G418-resistant clones were used.

g) Human fibroblasts

After surgical removal were skin biopsies in 4 ° C DMEM containing 10% FCS, 2 mM glutamine and Gentamycin, given. The biopsies were in one Tissue culture extensively with tweezers and surgical knife in laminar airflow in sterile 6 cm plastic dishes crushed. Then 3 ml of DMEM, containing 20% FCS, 2 mM glutamine and antibiotics Add and culture in a 37 ° C incubator given. After 10 days, the medium was replaced by DMEM, containing 10% FCS, exchanged. Then that became Medium continued to be changed twice a week. 4 weeks after Beginning of the culture, the cells were taken from the Tissue fragments were outgrown, trypsinized and for transfection into new culture dishes plated.  

An alternative, preferred method was the Skin pieces after crushing in fresh medium too Transfer and add 1 to 2 times with medium as needed to wash. 5 to 10 pieces of tissue were placed in a T25 Tissue culture flask whose surface area with DMEM plus 10% FCS had been wetted, given and even distributed, whereupon the bottle was turned over. This caused the biopsies to hang down ("hanging drop configuration ", this method was developed by Jones, 1989). After 1 to 3 h in the incubator the bottles were turned over again and with 1 to 2 ml Medium filled. Fixed biopsies were performed after 24 h made up to 5 ml; otherwise, the process became repeated. After 6 to 10 days, the first grew Fibroblasts dating from that time was once weekly changed the medium. Once the cells were confluent, they were in a T75 bottle passaged.

h) luciferase assay

The determination of luciferase activity was carried out as described in WO 93/07283.

i) cytokine ELISAs

The IL-6 ELISA was used by R Systems, the IL-8 ELISA purchased from Bender MedSystems.

j) LPS preparation

It became a commercially available LPS preparation Escherichia coli (0111: B4, Sigma). The The preparation was dissolved at 10 mg / ml in LPS-free water and prior to the preparation of serial dilutions in LPS sonicated for 5 minutes (SONOREX bath, 360  W). The final dilutions were made before the Use sonicated for 5 min.

example 1 Activation of NF-IL-6 and NF-κB by entry of adenovirus

There were stable clones of Pulmonary epithelial carcinoma cell line A549 produced as Reporter gene construct pNF-κB-luc or pIL-6-luc contained. With these two cell lines (below also as "A549 NF-κB cells" or "A549 IL-6 cells" referred) was the stimulatory behavior various agents tested in the following way: A defined number of cells were each plated (4 × 10⁴ cells per well of a 24-well plate for the A549 NF-κB cells; 4 × 10⁵ cells per well of a plate of 6 Wells for the A549 IL-6 cells) and 24 to 48 h later the test agents for 4 h in 2% Horse serum / DMEM exposed. The cells were then in given fresh medium (10% FCS / DMEM) and for the Luciferase analysis (triplicate) to the in Table 1 times (contents of 2 to 3 wells per sample). The control cells, too The same time harvested, the same Medium changes suspended with the difference that the Test agents were missing.

The activation behavior of each of these reporter cells as well as the concentration of the test substances is in Table 1 (in the treatment with Adenovirus / pL / DNA as a stimulus, the A549 NF-κB Cells with 20 μl transfection complex (4x10⁸  Virus particles corresponding to 10⁴ virus / cell) and the A549 IL-6 cells with 200 μl transfection complex treated, which is also 10⁴ viruses per cell corresponded).

To determine the time course of NF-IL-6 and NF-κB activation, the cells of the two cell lines, which were in the amounts indicated above in a 24-well plate and a 6-well plate, respectively, were each treated with 10⁴ adenovirus d11014 virus particles per cell (corresponding to 4 x 10⁸ viral particles for each well of the A549 NF-κB cells and 4 x 10⁹ virus particles for each well of the A549 IL-6 cells, respectively). The time course of induction is shown in Figure 1: the IL-6 promoter is fully activated after 12 hours and then rapidly returns to its initial level while the NF-κB promoter is activated and remains active for at least 50 hours. The latter finding is in contrast to the published activation profile of NF-κB itself, which, after its activation, returns to baseline levels after 6 to 12 hours due to the activation of IKB synthesis (Sun et al., 1993; Scott et al., 1993 Chiao et al., 1994). (One possible explanation for this is that the synthetic NF-κB promoter used lacks the appropriate inhibitory elements that ensure downregulation, while the IL-6 promoter, a natural sequence, has sequences that allow the normal control functions to function .)

Example 2 Blocking the activation of NF-κB by expression from VA1

In this example, it was investigated whether the introduction of a VAI gene blocks the activation of NF-κB, which is triggered by the entry of adenovirus / polylysine / DNA transfection complexes. For this purpose, 4 × 10⁴ A549 NF-κB test cells per well of a 24-well plate with 4 × 10⁸ psoralen / UV-inactivated adenoviruses d11014, corresponding to 10⁴ virus particles per cell, or with the corresponding number of viruses as part of the transfection complexes, corresponding to FIG μl Transfektionskomplex, treated and found that a similar activation of NF-κB is caused (up to 6 to 7 times after 72 h). The incorporation of E1B-encoding DNA into the transfection complex (500 μl of the complex mixture contained 6 μg of pCMVE1B DNA) had no effect on the inactivation of NF-κB. In contrast, expression of the adenovirus VA1 gene (caused by incorporation of VA1-encoding DNA into the transfection complex, 500 μl containing 6 μg of pXVAH) reduced activation to approximately 1.5 times the basal level ( Figure 2 complex means pSP65 , Complex / E1B means pCMVE1B, complex / VA1 means pXVAH).

Example 3 Blocking the activation of the IL-6 promoter by Expression of VA1

The promoter for the human IL-6 gene contains Binding sequences for the transcription factors NF-IL- 6 and NF-κB, which in activating this Promoter (Matsusaka et al., 1993; Betts et al., 1993).  

A549 IL-6 cells containing the luciferase gene under the control of the IL-6 promoter (NF-IL-6-luc) were used (each sample was in a well of a 6-well plate containing 4x each 10⁵ cells, duplicate determinations were made). In addition to the control changes in which only medium changes were made (sample 1), the cells were treated with PMA (phorbol myristyl acetate; 10 ^-8 M; sample 2), 10⁴ adenovirus particles contained in 200 μl transferrin-polylysine / DNA complex identified as DNA pSP65 contained (sample 3), or with 10⁴ virus particles contained in 200 ul TfpL / DNA complex containing as DNA pXVAH (sample 4), treated and after 10 h the luciferase activity as a measure of IL-6 activation measured. The result of the experiments is shown in FIG .

Example 4 Partial inhibition of by the entry of Adenovirus stimulated secretion of IL-6 and IL-8 in human dermal fibroblasts by expression of VA1

After the virus had been shown to stimulate both a NF-κB responsive promoter and the NF-κB / NF-IL-6 responsive IL-6 promoter (Table 1, Figures 1, 2 and 3), the production of IL-6 or IL-8 by transfected primary fibroblasts was analyzed as a measure of the activation of the two endogenous genes that respond to NF-κB / NF-IL-6.

Primary human fibroblasts (2 x 10⁴ cells per well) with either 10³ or 3 × 10³ Psoralen / UV-inactivated adenovirus d11014 particles  per cell, incorporated in StpL / TfpL / DNA Transfektionskomplexe, treated (this corresponded to 2 ul or 6 .mu.l transfection complex, the used DNA was either an empty plasmid (pSP65, Boehringer Mannheim) or the plasmid designated pXVAH, the contains the adenovirus VA1 sequence. The cells became 4 h with the transfection complexes in 2% Horse serum / DMEM treated, then became the medium removed and replaced by 10% FCS / DMEM. After 24 h (for IL-6) and 48 h (for IL-8) were aliquots of the Mediums removed and by ELISA on their content IL-6 and IL-8, respectively. It was found that the Treatment with the transfection complexes both the IL-6 as well as IL-8 secretion by these cells stimulated (Table 2). In all cases, the decreased Incorporation of the VA1 Gene into Transfection Complexes Amount of cytokine produced by about 35%. The initial stimulation of cytokine production (Virus entry) must take place before the gene with the Protective effect is introduced and into active RNA is transcribed. Therefore, measurements of the Cytokine production at later time points after Transfection to determine if the VA1 transfected cells at later time points, if higher concentrations of VA1 RNA have been formed are, a more pronounced suppression of Show cytokine production.

Example 5 Decrease of Gene Expression by Expression of Adenovirus genes E1A and E1A13S. Reinforcement of the Gene transfers by expression of E1B, E1B 19K and Bcl-2

First, a series of experiments were carried out to determine whether the expression of certain adenovirus genes is capable of enhancing gene expression after transferrin infection. A defined amount of the luciferase expression plasmid pCMVL (5 μg) was mixed with various plasmid DNA (1 μg each), incorporated into TfpL / StpL / adenovirus transfection complexes (total volume 500 μl) and applied to primary human fibroblasts (2 × 10⁴ cells per well of a 24-well plate add 50 μl of complex, corresponding to 2.5 × 10⁴ virus particles per cell, using triplicate determinations In Figure 4, "minus" means pSP65, the test DNA is indicated respectively). The obtained luciferase activity was measured 48 to 72 hours after transfection. Incorporation of purified total DNA from the E4-negative, E1-positive adenovirus d11014 produced a twofold increase in gene expression ( Figure 4). It was believed that the enhancement was due to E1 expression. Subsequently, an E1 expression plasmid (pE1pAALM) was introduced into the cells together with the reporter plasmid and found that this also causes an increase in gene expression (3-fold). The E1 region contains two major gene functions, E1A and E1B. (The E1A products have the effect of separating E2F from the Rb protein, on the other hand they modulate the transcriptional activity of many cellular and adenovirus promoters.) The major transcriptional transactivator function is contained in the 13S gene product The 1B gene encodes two major functions: the E1B 55K gene product binds p53 and alters its function The 19K E1B gene product is thought to act below p53 to block the apoptotic response.)

Co-expression of plasmids was tested either the complete E1A or the complete E1B Region under the control of the CMV promoter. The E1B plasmid gave a 7 fold amplification of Gene expression. In contrast, the E1A Plasmid inhibition of gene expression (the 0.3 times the control). Also called the 19K E1B gene an increase in gene expression (8.6fold).

The E1B 19K protein has recently been shown to be a potent analogue of the anti-apoptotic gene Bcl-2 is (Rao et al., 1992). It therefore became Bcl-2 also tested and found that it was a 5.5-fold increase in gene expression causes.

Example 6 Examination of toxicity for specificity

Next, it was tested whether the decrease of Gene expression on non-specific toxicity caused by the high virus / cell ratios is, is due (the experiments were with approx. 50,000 virus particles per cell performed).

For this transfection complexes containing pCMVL plus the plasmid pSP65, or the plasmids containing the sequence for Bcl-2, E1A or E1B 19K, at virus particle counts of 3 × 10⁴ ( Fig. 5A), 10⁴ ( Fig. 5B) or 3 × 10 3 ( Figure 5C) per cell on primary fibroblasts and luciferase activity measured over time. The transfections were performed as described in the previous examples, with the cells being given 6, 18 and 60 μl transfection complexes per well containing 2x10⁴ fibroblasts. The result of the experiments is shown in FIG. 5: Similar gene expression patterns as in the preceding examples (decrease in E1A-treated cells, amplification in 19K and Bcl-2-treated cells, respectively) were found, irrespective of the virus dose used ,

In the previous example, even with the 19K or Bcl-2 transfected cells to loss Expression activity at later times, especially after 6 days, shown. It was therefore in Considered, if not a second phenomenon, that in Relationship with the inflammatory response stands, like she has shown in the previous example, the Health of transfected culture affected. If the transfected cells due to the Transfektionsvorganges an activation of Immune response could show the secretion of cytokines or toxins from the cells' life expectancy Restrict culture.

In order to investigate this phenomenon, experiments were performed at the time of transfection using transfection complexes containing 5 μg pCMVL plus 1 μg pSP65 or 1 μg of the plasmid containing E1A, E1A 13S, E1B 19K or Bcl-2. (In these experiments, the cells of one well received 18 μl of transfection complex.) From day 10, the culture medium was replaced daily with fresh medium; This measure was based on the consideration that the medium change would lower the concentrations of the toxins. The result of these experiments is shown in Figure 6: It was found that the chosen procedure enhanced the long-term gene expression of the cultures when pSP65, Bcl-2 or 19K plasmids were used. In contrast, the expression of the E1A and E1A 13S cultures decreased to a similar extent as in the non-washed cultures of the previous experiments. The presence of 19K still showed a one-decade improvement in gene expression after 21 days, indicating a role for apoptosis.

Example 7 Investigation of the morphology of transfected cells

Primary human fibroblasts were transfected with the reporter plasmid pCMVβgal plus pSP65 or plus pCMV19K (2 × 10⁴ fibroblasts per well of a 24-well plate received 18 μl transfection complex; 500 μl complex contained either 4 μg pCMVL plus 2 μg pSP65 or 4 μg pCMVL plus 2 μg pCMV19K). 48 h after transfection, the cells were stained with β-gal as described in WO 93/07283 to study the morphology of the cells that successfully ingested and expressed the transfected DNA. It was found that at this early stage after transfection the two cell populations express the transfected cells within an order of magnitude. However, while the β-gal expressing cells in the absence of pCMV19K show a predominantly rounded and partially detached morphology ( Figure 7, left panel) that matches an apoptotic phenotype, the cells transfected with pCMV19K co-exhibited a predominantly flat and adherent morphology ( Fig. 7, right panel). The altered morphology is likely to be related to the more extensive and increased gene expression obtained with 19K.

Example 8 Inhibition of adenovirus or LPS induced toxicity by pre-expression of E1B 19K and Bcl-2

In the course of preliminary experiments, in which the cells LPS in Presence of adenovirus had been exposed found that the rapidly occurring toxicity in These cells have a morphology of dying cells which is similar to apoptosis (e.g. fragmented nuclei, condensation). On it was believed that the penetration of LPS into the cell during the onset of adenovirus an apoptotic Answer causes.

To investigate this phenomenon, transfection complexes containing LPS plus either E1B 19K gene or Bcl-2 gene were prepared to determine if the total introduction of the apoptosis inducer LPS with the anti-apoptosis gene Bcl-2 would block cell death. It has been found that there is no reduction in toxicity, presumably because the rate of the toxic response, which becomes noticeable 4 hours after treatment, does not allow sufficient 19K or Bcl-2 gene product to accumulate. Based on this observation, an alternative method for determining whether 19K or Bcl-2 could provide protection against LPS-induced toxicity was developed: Stable cell lines carrying either the 19K or Bcl-2 gene were identified LPS / adenovirus complexes brought into contact. For this purpose, A549 cells were plated as control cells and A549 19K cells and A549 Bcl-2 cells at 4 × 10⁴ cells per well of a 24-well plate and with 50 .mu.l transfection complex containing 6 .mu.g pCMVL per 100 ul complex and either 0, 10 or 100 ng LPS per 6 μg DNA. The expectation that the preceding expression of each of these proteins should have a protective effect was met: As shown in Figure 8, control cells exposed to the adenovirus / pL / DNA complexes became a function of increasing LPS content in the sample killed (see sample 1: no LPS, with samples 2 and 3: 10 and 100 ng LPS / 6pg DNA). In cells expressing either 19K or Bcl-2, a reduction in toxicity occurred to 39% (sample 2) and 77 or 60%, respectively (samples 5 or 8, 19K or Bcl-2). In evaluating these results, it should be noted that the cells evaluated in this experiment were still pools of stably expressing cells; of pure populations of 19K or Bcl-2 expressing cells, it is expected that a higher protective effect (up to 100%) will be obtained.

Example 9 Enhancement of gene transfer by glucocorticoid Dexamethasone alone or in combination with an anti- Apoptosis gene

Primary human fibroblasts were used Adenovirus / polylysine / DNA complexes are transfected as described in Example 5, using as DNA either pCMVL / pSP65 or pCMVL / pCMV19K in the ratio of each 5: 1 was used. 3 hours after transfection samples were taken in fresh 10% FCS / DMEM medium or in 10% FCS / DMEM medium containing 3 μM dexamethasone (Sigma), convicted. 24 h after transfection were the samples (double determination) were harvested and the  Luciferase activity determined. It turned out that the Presence of dexamethasone in the absence of pCMV19K increased luciferase expression by a factor of 1.65. In the presence of both dexamethasone and pCMV19K the gain was 2.71x (Table 3).

conditions fold induction PCMVL / pSP65 1.0 PCMVL / pSP65 / dexamethasone 1.65 PCMVL / pCMV19K 1.0 PCMVL / pCMV19K / dexamethasone 2.71

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Claims (17)

1. A method for introducing foreign material, in particular nucleic acid, into higher eukaryotic cells, characterized in that in the cell except the foreign material and the transfection components

• a) introduces into the cell one or more nucleic acid molecules, in particular DNA molecules, whose expression products at least partially block the apoptosis of the host cell triggered by the introduction of the foreign material, and / or that one
• b) at least partially inhibits the inflammatory response of the cells by
  • i) the cells externally treated with one or more anti-inflammatory substance (s) and / or
  • ii) introducing into the cells one or more anti-inflammatory substance (s) or the nucleic acid, in particular DNA, molecules coding for it.

2. The method according to claim 1, characterized that the foreign material is DNA, which with a, optionally with a ligand for the Target cell conjugated, polycation complexed is, and that the DNA complex in the presence of Adenovirus or adenovirus-polycation conjugate in the cells are introduced. 3. The method according to claim 1 or 2, characterized characterized in that the DNA defined in a) Molecule with anti-apoptosis activity for human Bcl 2 coded.   4. The method according to claim 1 or 2, characterized characterized in that the DNA defined in a) Molecule with anti-apoptosis effect for adenovirus E1B 19K coded. 5. The method according to claim 1 or 2, characterized characterized in that the DNA molecule is labeled with anti- Apoptosis effect codes for a gene product that p53 inactivated. 6. The method according to any one of claims 2 to 5, characterized in that the DNA molecule with anti-apoptosis effect in common with in the Cell to be introduced as part of a DNA DNA-polycation complex is present. 7. The method according to claim 6, characterized that the DNA to be introduced into the cell and the DNA molecule with anti-apoptosis effect on separate plasmids are present. 8. The method according to claim 6, characterized that the DNA to be introduced into the cell and the DNA molecule with anti-apoptotic effect in common present on a plasmid. 9. The method according to claim 1, characterized that the anti-inflammatory substance adenovirus VA1 is. 10. The method according to claim 1 or 9, characterized characterized in that the anti-inflammatory Substance in the form of the coding DNA in the Cell is introduced.   11. A method according to claim 2, 9 and 10, characterized characterized in that the VA1-DNA together with the to be introduced into the cell DNA as a component a DNA-polycation complex is present. 12. The method according to claim 11, characterized characterized in that the complex also contains the DNA Contains molecule. 13. The method according to claim 11 or 12, that the two or the three DNA molecules on separate Plasmids present. 14. The method according to claim 11 or 12, that the two or the three DNA molecules together on one Plasmid present. 15. The method according to claim 1, characterized that a DNA molecule with anti- Apoptosis effect and the cells externally with an anti-inflammatory effect Substance treated. 16. The method according to claim 15, characterized characterized in that the anti-inflammatory Substance is a glucocorticoid. 17. transfection complex containing DNA with one, optionally with a ligand for the Target cell conjugated, polycation complexed as well as adenovirus or an adenovirus Polycation conjugate, characterized in that he also has a DNA molecule with anti-apoptosis Action and / or a DNA molecule coding for a substance with an anti-inflammatory effect, contains.