WO1995033062A2 - Verfahren zum einbringen von fremdmaterial in höhere eukaryotische zellen - Google Patents
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- WO1995033062A2 WO1995033062A2 PCT/EP1995/001989 EP9501989W WO9533062A2 WO 1995033062 A2 WO1995033062 A2 WO 1995033062A2 EP 9501989 W EP9501989 W EP 9501989W WO 9533062 A2 WO9533062 A2 WO 9533062A2
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- C07K14/52—Cytokines; Lymphokines; Interferons
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- C12N2710/10011—Adenoviridae
- C12N2710/10211—Aviadenovirus, e.g. fowl adenovirus A
- C12N2710/10222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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- C12N2710/00011—Details
- C12N2710/10011—Adenoviridae
- C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
- C12N2710/10322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
Definitions
- the present invention relates to a
- Standard methods for transfecting cells include Calcium phosphate (for in vitro applications), cationic lipids (Feigner et al., 1993) or liposomes.
- Vector systems for the transfer of genes into the cell (Wilson et al., 1990; Kasid et al., 1990, WO 93/03769).
- Adenoviruses or fusogenic peptides are used to increase the efficiency of the gene transfer by breaking down the DNA complexes internalized into the cell in the
- Lysosomes are 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;
- Adenovirus-polylysine conjugates can be conjugated with transferrin-polylysine conjugates with DNA
- Adenovirus mutants suggest that host responses are very early, possibly even before viral gene expression.
- One component of this host response is one
- interferon responsive genes genes that respond to interferon
- ISGF3 interferon responsive transcription factor
- Binding sites are located upstream of a series of genes responsive to interferon.
- One of the activated genes is the protein kinase p68, which is activated by double-stranded RNA.
- p68 is synthesized in an inactive form and is autocatalytic in the presence of dsRNA
- NF-KB has also been reported to be activated by dsRNA
- p68 may directly phosphorylate IKB and activate NF- ⁇ B.
- the type C adenoviruses have two strong mechanisms to address 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 which can bind to the p68 kinase without activating the kinase and which can prevent binding and activation by the actual activators (Manche et al., 1992;
- Another inflammatory reaction mechanism as a result of the entry of virus is likely to be the 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, often in conjunction with NF- ⁇ B, activates the IL-6 gene itself as well as a number of other inflammatory response genes such as TNF and IL-1.
- IRF 1 Interferon Response Factor 1
- Adenovirus ensures the triggering of the virus gene expression cascade.
- Another line of defense of the host cell is the apoptotic response. It has long been known that mutations that map in the EIB 19K region for the deg phenotype, an enhanced cytopathic effect that is accompanied by degradation of the host's chromosomal DNA (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). Younger ones
- Virus infection can be (Levine et al., 1993): In the absence of Bcl-2, the cell undergoes apoptosis, which limits virus production to a short, acute phase. Chronic virus production takes place in the presence of Bei-2 expression because the apoptotic response to the acute phase infection is absent. Anti-apoptotic activities were also undertaken in the Epstein-Barr virus (the BHER1 gene; Pearson et al.,, 1987), in the baculovirus (the p35 gene; Sugimoto et al., 1994) and in the African swine fever virus (the LMW5-HL gene; Neilan et al ., 1993). This indicates that the apoptotic response to the virus infection occurs in many cases and virus strategies have been developed to block this response.
- the viral capsid acts only as a reagent that breaks the endosomes, which is a function of the surface proteins of the viral capsid. Inactivation methods have been developed which
- LPS lipopolysaccharide
- the object of the present invention was to elucidate the mechanisms involved in the
- the present invention relates to a method for treating higher eukaryotic cells
- a) introduces one or more nucleic acid molecules, in particular DNA molecules, into the cells, the expression products of which by introducing the
- Block at least partially apoptosis of the host cell triggered by foreign material, and / or that b) at least partially inhibit the inflammatory response of the cells by
- the cells are treated externally with one or more anti-inflammatory substance (s) and / or
- DNA molecules encoding nucleic acid, especially DNA molecules.
- the DNA molecules defined in a) are referred to below as “anti-apoptosis genes” or as “DNA molecules with anti-apoptosis activity”.
- the cells in addition to components a) and optionally b) ii)
- apoptosis encompasses all toxic effects which are attributable to a) direct activation of the apoptotic response; b) activation of reactive oxygen metabolites which directly or indirectly induce apoptosis; c) Activation of the secretion of TNF, IL-6 or another cytokine, which the
- Adenovirus that is introduced into the cell. This
- Toxicity effects occur are applied, in particular to gene transfer methods, e.g. when using recombinant adenovirus vectors.
- gene transfer methods are used in vitro and in gene therapy both in vivo and ex vivo, in particular in fibroblasts, hematopoietic cells, endothelial cells or lung epithelial cells.
- the use of the method according to the invention can also include all applications in the treatment of eukaryotic cells in which one
- the transplanting cells can prevent apoptosis.
- the anti-apoptosis gene is the human Bcl-2 gene (Seto et al., 1988).
- the anti-apoptosis gene is the EIB 19K gene of adenovirus type 2 or 5 (White and Cipriani, 1990).
- Anti-apoptosis genes suitable for the invention are the BHRF1 gene of the Epstein-Barr virus (Pearson et al., 1987), the baculovirus genes p35 (Sugimoto et al., 1994) and iap (Clem and Miller, 1994), the LMW5- HL gene from Epstein-Barr virus (Pearson et al., 1987), the baculovirus genes p35 (Sugimoto et al., 1994) and iap (Clem and Miller, 1994), the LMW5- HL gene from Epstein-Barr virus (Pearson et al., 1987), the baculovirus genes p35 (Sugimoto et al., 1994) and iap (Clem and Miller, 1994), the LMW5- HL gene from Epstein-Barr virus (Pearson et al., 1987), the baculovirus genes p35 (Sugimoto et al., 1994) and ia
- African swine fever virus (Neilan et al., 1993) and the ced-9 gene from Caenorhabditis elegans (Sugimoto et al .; 1994).
- proteases involved proteins similar to Bei-2 and Bei-2 are believed to block activation of the proteases designated ICE or ICE-like. Since these protease (s) obviously have a crucial function for apoptosis, their blocking also plays a crucial role.
- a component which acts further upstream in the apoptosis signal transmission path is, for example, p53, the activation of which triggers one of the signal transmission paths which lead to apoptosis.
- genes that inactivate p53 are EIB 55K from adenovirus type 2 or type 5 (White and Cipriani, 1989) or El functions from other strains of adenovirus, e.g. Adenovirus 12 or from CELO virus (Chick Embryo Lethal Orphan Virus), the E6 gene from human
- Papillomavirus-16 (Levine, 1990), SV 40's large T antigen (McCarthy et al., 1994), the mdm-2 gene
- Mammalian cells e.g. Fibroblasts, with a
- Reporter plasmid e.g. a luciferase plasmid to transfect and monitor expression of the reporter gene over a period of time.
- test DNA e.g. genomic virus DNA fragments or DNA molecules from a cDNA library
- suitable expression vectors e.g.
- the cells are optionally additionally transfected with a DNA sequence which is known to be the
- the anti-apoptotic effect of a test DNA sequence is shown by the fact that when the cells are co-transfected with it Sequence the expression of the reporter gene is higher than when transfecting the cells with the reporter gene alone.
- the effect of the test gene can be compared with that of the known gene.
- an anti-apoptotic DNA molecule of the chicken adenovirus type I was first used in the context of the present invention
- CELO Chicken Embryo Lethal Orphan
- This DNA molecule lies on a Smal / Hindlll fragment which is located in the CELO virus genome in the range between approximately 84.3 to approximately 88.7 mapping units.
- the nucleotide sequence shown in FIG. 14 was determined from the Smal / HindiII fragment isolated in the context of the present invention (SEQ ID NO: 1).
- the Smal / HindiII fragment was obtained from an EcoRI fragment called 9R1, which ranged from 81.2 to 92.7 mapping units
- all of the CELO virus fragments obtained which have the anti-apoptotic effect such as e.g. the
- the EcoRI fragment 9R1 of which the Smal / HindIII fragment is a partial sequence, can be used as a whole.
- a further possibility consists in the HindiII fragment of the located between mapping units 77.8 and 88.7 on the CELO virus genome
- Fragments 9R1 and 7H3 have an 80% overlap area that
- the size of the DNA molecule in the area or in the vicinity of the specified fragment areas is not critical.
- the DNA which essentially consists only of the open reading frame, is preferably used.
- This DNA can optionally be shortened to the sections responsible for the anti-apoptotic function, or mutants can be constructed with a view to enhancing the activity.
- the selection of suitable mutants can be carried out by testing correspondingly mutated DNA sequences, as described in the examples, for their ability to increase gene expression.
- Reading frame is by screening the mutated DNA sections for anti-apoptotic effects. A number of deletion mutants were constructed for this. It turned out that all those constructions by which
- GAM-1 the amino acid sequence is given in SEQ ID NO: 2 also showed a series of seven leucine residues. This motif is known to fold into a structure (called “leucine zipper”) that is often a site of protein-protein interactions (Busch and Sassone-Corsi, 1990; Lumb and Kim, 1995) assumed that the
- Leucine-zipper dimersization domain works as an alpha-helical structure, the introduction of amino acids such as proline, which destroy alpha-helices, can thus impair zipper dimerization. It was
- GAM-1 therefore either functions as a homodimer or there are other binding partners for GAM-1.
- GAM-1 has no homologies with any of the known anti-apoptical proteins, including members of the Bcl-2 family, adenovirus 5 EIB 19K, the Bax family, the baculovirus IAP family, the
- Nematode protein Ced-3 (Furthermore, the complete sequence of the CELO virus genome showed that the CELO virus has no genes that are homologous to the Ad2 / 5 EIB 19K region. It could therefore be that the CELO virus has no EIB 19K region and hence the GAM-1 gene function used as an anti-apoptotic gene.)
- the invention thus relates to a DNA molecule containing the ones in FIG. 14 or SEQ ID NO: 1 shown nucleotide sequence or a partial sequence thereof coding for a functional gene product with anti-apoptotic effect.
- a DNA molecule with the sequence of the open one shown in FIG. 14 or SEQ ID N0: 1 is preferred
- Reading frame including degenerate variants of it, or mutants for a functional
- the invention relates to those derived from the DNA molecules according to the invention
- Polypeptides in particular the protein of the designation GAM-1 (SEQ ID NO: 2) encoded by the open reading frame, and functional derivatives thereof.
- Mechanism may be one
- Fibroblasts were transfected on the one hand with a luciferase reporter gene and on the other hand with test plasmids which contained different fragments of GAM-1. Two days later, the cells were trypsinized and a defined number of cells were plated on polyHEMA-coated cell culture plates. Seven days later, the adherent and non-adherent cells were harvested and examined for luciferase activity. It was found that the
- Plasmid encoding EIB 19 K, Bcl-2 or GAM-1 transfected Two days after the transfection, a defined number of cells were placed on a series of cell culture plates with a PolyHEMA coating
- Bei-2 should have a stronger effect than EIB 19 K or GAM-1; the one obtained without PolyHEMA
- Luciferase expression remained unchanged at Bei-2 even at the highest PolyHEMA concentrations.
- Identified DNA molecules coding for GAM-1 can also be mutated if the mutations either do not result in a change in the amino acid sequence or the changes do not affect any amino acids which are essential for the function.
- Transfer infection is based on the anti-apoptosis gene simply in the form of a plasmid together with the plasmid containing the DNA sequence, the expression of which in the cell is said to achieve the desired effect (hereinafter referred to as "effective DNA”), for example a
- Adenovirus polylysine The molar ratio of active gene and anti-apoptosis gene can be determined using
- Titration can be determined by measuring the expression of the active gene at different proportions of the anti-apoptosis gene under otherwise identical conditions over a longer period of time. In the context of the present invention, a ratio of 5: 1 was chosen, which has proven to be favorable. Since the gene products of some anti-apoptosis genes show a very strong effect, smaller proportions can also be used. An upper limit due to any toxicity can also be determined by titration.
- the molar ratio of the two genes is 1: 1 if they are each present on the plasmid in a copy .
- transcription factors namely NF-IL-6 and NF- ⁇ B. This created the prerequisite for activating these transcription factors in response to the occurrence of adenovirus or
- Substances come, for example, treatment with retinoic acid ("Retinoic acid”) or other retinoids, which have been reported to inhibit IL-1-induced IL-6 production by lung fibroblasts (Gross et al., 1993; Zitnik et al., 1994).
- Retinoic acid retinoic acid
- other retinoids which have been reported to inhibit IL-1-induced IL-6 production by lung fibroblasts (Gross et al., 1993; Zitnik et al., 1994).
- Mode of action of this class of substances is likely to be that they control the concentration of the IL-6 mRNA.
- Another class of substances for inhibiting the inflammatory host response are:
- Glucocorticoids that have been shown to inhibit the production of IL-6 and / or IL-8 in response to inflammatory stimuli (Ray et al., 1990; Barber et al., 1993; Wertheim et al., 1993).
- Dexamethasone acts on the activation of the enzyme
- This enzyme is used in response to numerous other organs.
- Another group of anti-inflammatory substances that can be used in the context of the present invention are inhibitors of arachidonic acid metabolism.
- Arachidonic acid is metabolized to proinflammatory prostaglandins and thromboxanes via the cyclooxygenase pathway. This path, which is shown schematically in Fig. 12, can be done by the
- Cyclooxygenase inhibitors aspirin acetylsalicylic acid
- Ibuprofen acetylsalicylic acid
- indomethacin Flower et al., 1985
- Arachidonic acid runs through the 5-lipoxygenase processing, whereby leukotrienes are formed.
- 5-Lipoxygenase can be obtained by NDGA (Nordihydroguaiaretic Acid; Burkart and Kolb, 1993; Cifone et al., 1993;
- the cells can also be treated with anti-inflammatory polypeptides such as IL-10 or TGF- ⁇ .
- anti-inflammatory polypeptides such as IL-10 or TGF- ⁇ .
- the anti-inflammatory substance is expediently added to the medium.
- step b) ii) the anti-inflammatory substance as such or in a preferred one
- Embodiment in the form of the DNA coding therefor introduced into the cell e.g. in the form of a plasmid which contains a sequence coding for an anti-inflammatory protein such as IL-10 (Moore et al., 1990) or TGF- ⁇ (Massague et al., 1987).
- an anti-inflammatory protein such as IL-10 (Moore et al., 1990) or TGF- ⁇ (Massague et al., 1987).
- a preferred anti-inflammatory substance used in the context of the present invention is VA1.
- Adenovirus VA1-RNA is a small RNA molecule synthesized by RNA polymerase III, which is necessary for the efficient expression of the virus genome
- Transfection can be used to modulate the activation of NF- ⁇ B that is triggered by the transfection process.
- VA1 expression inhibits the activation of inflammatory cytokines may be due to a phenomenon that is independent of the increase in the stability of the mRNA or its amount, as reported for the calcium phosphate method. Inhibition of p68 kinase activation may play a key role in this phenomenon.
- the VA1 can be imported into the cell as an RNA molecule or, in a preferred embodiment, in the form of the VA1 DNA.
- Transfection complexes are integrated. It is also possible to combine two of the three sequences, or all three, on a single plasmid.
- a DNA containing the factor VIII sequence as therapeutically effective DNA an anti-apoptosis gene such as in-2 or EIB 19K and the VA1 DNA.
- genes can be used which have an effect corresponding to VA1, e.g. EBER of the Epstein Barr virus (Clarke et al., 1990; Clarke et al., 1991) or the TAR-RNA from the HIV virus (Gunnery et al., 1990).
- the invention relates to a transfection complex containing a nucleic acid molecule to be expressed in the cell, which is complexed with a polycation, optionally conjugated with a ligand for the target cell, and
- Adenovirus or an adenovirus-polycation conjugate wherein the complex also contains a DNA molecule with anti-apoptosis activity and / or a DNA molecule coding for a substance with anti-inflammatory activity.
- the invention also relates to a
- compositions containing such a transfection complex in which the nucleic acid molecule to be expressed in the cell is a therapeutically or gene-therapeutically effective DNA molecule.
- the pharmaceutical preparation contains the usual additives, such as nutrients, etc., for use on cells.
- Fig. 1 Time course of activation of NF-IL-6 and
- Fig. 2 Blocking the activation of NF-KB by
- Fig. 3 Blocking the activation of the IL-6 promoter by expression of VA1
- FIG. 6 Influence of the expression of Bcl-2, EIB 19K, EIA and EIA 13S on the long-term gene expression.
- FIG. 7 Influence of the transfection on the morphology of fibroblasts
- Fig. 8 Inhibition of adenovirus or LPS
- Fig. 11 Effect of dexamethasone, ibuprofen or
- Fig. 12 Comparison of the enhancement of gene expression by different inhibitors of
- Fig. 13 Restriction map of the CELO virus genome
- Fig. 16 Anti-apoptotic effect of various CELO virus fragments
- Fig. 17 Open reading frame of the EcoRI fragment from
- Fig. 18 Assignment of the anti-apoptotic effect to the correct reading frame. Comparison of different
- Fig. 19 Influence of the mutation of the leucine zipper on the anti-apoptotic effect
- Fig. 20 Effect of the CELO virus gene on the
- Fig. 21 Comparison of the effect of different anti-apoptotic genes on the
- Fig. 22 Effect of combinations of different genes with anti-apoptotic effects
- adenovirus type 5 (nucleotide 1-5778) was obtained by digesting the adenovirus dll014 DNA (Bridge and Ketner, 1989) with Xhol plus Asel. This fragment was made with Klenow Polymerase treated and in the Smal site of the
- Plasmids pAALM obtained from pSP64 (Boehringer
- the VA1 expression plasmid pXVAH was constructed by inserting the 5324 bp HindII fragment (nucleotide 6241-11656) of the adenovirus DNA into the HindIII site of pAALM to obtain pHVAH. pHVAH was then cleaved with XBal and religated to obtain pXVAH (this made a large piece from the XBal site in pAALM to the XBal site
- the clone pXVAH obtained contained the adenovirus sequence of nucleotide 10589-11565. ii) Luciferase reporter plasmid pNF- ⁇ B-Luc
- the plasmid pNF- ⁇ B-Luc responsive to NF- ⁇ B was developed as a derivative of that described by Boehmelt et al., 1992,
- plasmid pTK3kbB described constructed. To do this, pTK3kbB was cut with Xhol and Ncol to remove most of the sequence coding for CAT with
- pCMVE1A The expression plasmids named pCMVE1A, pCMVE1B and pCMV19K were from White and
- the expression plasmid designated pCMV-Bcl-2, was prepared by the human Bcl-2 cDNA, flanked by two EcoRI sites (Seto et al., 1988), downstream from a CMV immediate early promoter in the
- Plasmid DNA pCMVL The construction of the plasmid is described in WO 93/07283. viii) plasmid DNA pCMVßgal
- the E4-deficient adenovirus 5, dll014 (Bridge and Ketner, 1989) was grown in the complementing cell line W162 (Weinberg and Ketner, 1983). Pellets from infected cells became 2 ml / 2 x 10 7 cells in 20 mM HEPES, pH 7.4, 1 mM PMSF
- the opalescent virus bands (either 1.31 g / cm 3 immature or 1.34 g / cm 3 mature) were harvested. The biotinylation of the obtained
- Glycerin was carried out as described in WO 93/07283 or by Wagner et al., 1992, and Cotten et al., 1992, described.
- the virus samples were quantitatively determined via the protein concentration (Biorad Bradford assay with BSA as standard), the
- Samples of biotinylated, psoralen / UV inactivated adenovirus d11014 (8 ⁇ l, 1 ⁇ 10 12 particles / ml) were diluted in 150 ⁇ l HBS and mixed with 1 ⁇ g StpL in 150 ⁇ g HBS for 30 min at room temperature. Aliquots of 6 ⁇ g plasmid DNA (in the case of mixtures of pCMVL-DNA and another plasmid the ratio was 5: 1) were diluted in 100 ⁇ l HBS and then with the
- the human lung carcinoma cell line A549 (ATCC CCL 185) was used for the production of the clonal cell lines containing the reporter plasmid pNF- ⁇ B-Luc or pIL-6-Luc.
- the plasmids were co-transfected with the neomycin phosphotransferase expression plasmid called pMuatt (a pUCl9 plasmid containing the TKneo sequence; Grosveld et al., 1982) using the Transfeetam method (Behr et al., 1989) selected for cells that are resistant to 400 ⁇ g / ml G418 (Sigma). Clonal derivatives that expressed luciferase after induction by PMA were identified and expanded.
- A549 cells containing the expression plasmid for Bcl-2 and EIB 19K were obtained in a similar manner, with the difference that the
- Neomycin phosphotransferase plasmid pSVneo (Clontech) instead of pMuatt and that pools of G418-resistant clones were used instead of pure populations.
- pSVneo Neomycin phosphotransferase plasmid pSVneo
- Glutamine and antibiotics were added and the culture placed in a 37oC incubator. After 10 days it was Medium replaced by DMEM containing 10% FCS. Then the medium was continued twice a week
- An alternative, preferred method was to transfer the pieces of skin into fresh medium after comminution and to wash them with medium once or twice as required.
- 5 to 10 pieces of tissue were placed in a T25 tissue culture bottle, the surface of which had been wetted with DMEM plus 10% FCS, and distributed evenly, whereupon the bottle was turned over. This caused the biopsies to hang ("hanging drop configuration"; this method was described by Jones, 1989).
- the bottles were turned over again and filled with 1 to 2 ml of medium. Fixed biopsies were filled up to 5 ml after 24 h; otherwise the process was repeated.
- the first fibroblasts grew out after 6 to 10 days, from this point on the medium was changed once a week. Once the cells were confluent, they were placed in a T75 bottle
- the plates were coated with PolyHEMA (poly (2-hydroxyethyl methacrylate) by adding 200 ⁇ l of a PolyHEMA solution (Sigma, Cat. No. P-3932; 10 mg / ml in 95%
- Culture plates used were prepared by a method similar to that described by Folkman and Moscona, 1978. For this purpose, normal tissue culture plates (24-well plates) with 200 ⁇ l per well were used
- the IL-6 ELISA was obtained from R&D Systems, the IL-8 ELISA from Bender MedSystems. k) LPS preparation
- the preparation was dissolved in 10 mg / ml in LPS-free water and sonicated in LPS-free water for 5 minutes before making serial dilutions (SONOREX bath,
- Lung epithelial carcinoma cell line A549 generated which as a reporter gene construct pNF-KB-luc or pIL-6-luc
- Adenovirus / pL / DNA as a stimulus were the A549 NF- ⁇ B cells with 20 ⁇ l transfection complex (4 ⁇ 10 8)
- Virus particles corresponding to 10 4 viruses / cell and the A549 IL-6 cells with 200 ⁇ l transfection complex treated, which is also 10 4 viruses per cell
- the time course of the induction is shown in FIG. 1: The IL-6 promoter is fully activated after 12 h and then quickly returns to its initial level while the NF- ⁇ B promoter is activated and
- Adenovirus / polylysine / DNA is triggered blocked. For this, 4 x 10 4 A549 NF- ⁇ B test cells per
- Adenovirus VA1 gene (caused by incorporation of the DNA coding for VA1 in the
- Transfection complex 500 ⁇ l contained 6 ⁇ g pXVAH activation to approx. 1.5 times the basic value (FIG. 2; complex means pSP65, complex / EIB means pCMVE1B, complex / VA1 means pXVAH).
- the promoter for the human IL-6 gene contains
- NF-IL-6-luc Contained luciferase gene under the control of the IL-6 promoter (NF-IL-6-luc) (each sample was in a well of a 6-well plate, each containing 4 ⁇ 10 5 cells. Duplicate determinations were made
- the cells were contained in 200 ⁇ l with PMA (phorbol myristyl acetate; 10 -8 M; sample 2), 10 4 adenovirus particles
- Transferrin-polylysine / DNA complex which contained pSP65 as DNA (sample 3) or with 10 4 virus particles,
- Luciferase activity measured as a measure of IL-6 activation. The result of the tests is shown in Fig. 3.
- Psoralen / UV inactivated adenovirus d11014 particles treated per cell incorporated in StpL / TfpL / DNA transfection complexes (this corresponded to 2 ⁇ l or 6 ⁇ l transfection complex, the DNA used being either an empty plasmid (pSP65; Boehringer Mannheim) or the plasmid with the designation pXVAH, which is the adenovirus VA1
- the cells were washed with the transfection complexes in 2% for 4 h
- Fibroblasts were applied (50 ⁇ l of complex, corresponding to 2.5 ⁇ 10 4 virus particles per cell, were applied to 2 ⁇ 10 4 cells per well of a 24-well plate, whereby triple determinations were carried out.
- “minus” means pSP65; Test DNA is labeled).
- Luciferase activity was 48 to 72 h after
- the E1 region contains two main gene functions, EIA and E1B.
- the E1A products have the effect of separating E2F from the Rb protein, on the other hand they modulate the transcription activity of many cellular and adenovirus promoters
- the main transcriptional transactivator function is contained in the 13S gene product.
- the 1B gene codes for two main functions: the EIB 55K gene product binds p53 and changes its function.
- the 19K E1B gene product is believed to act below p53 to block the apoptotic response.)
- the co-expression of plasmids which carried either the complete EIA or the complete E1B region under the control of the CMV promoter was tested.
- the E1B plasmid showed a 7-fold amplification of the
- the 19K E1B gene also caused an increase in gene expression (8.6-fold).
- the E1B 19K protein has recently been shown to be a potent analog of the anti-apoptotic gene Bcl-2 (Rao et al., 1992). Bcl-2 was therefore also tested and found to be a
- Gene expression is due to a non-specific toxicity caused by the high virus / cell ratio (the experiments were carried out with approximately 50,000 virus particles per cell).
- transfection complexes containing pCMVL plus the plasmid pSP65, or the plasmids which contained the sequence for Bcl-2, E1A or E1B 19K (the amounts of reporter plasmid pCMVL and respective
- Test plasmid were, as in the previous example, 5 ⁇ g and 1 ⁇ g, for virus particle numbers of 3 ⁇ 10 4 (FIG. 5A), 10 4 (FIG. 5B) or 3 ⁇ 10 3 (FIG. 5C) per cell on primary Fibroblasts applied and the
- Luciferase activity measured over time. The transfections were carried out as in the previous examples described, the cells 6, 18 and 60 ul transfection complex per
- Enhancement in 19K or Bcl-2-treated cells was found, regardless of the virus dose used.
- Transfection complexes containing 5 ⁇ g pCMVL plus 1 ⁇ g pSP65 or 1 ⁇ g of a plasmid containing E1A, E1A 13S, E1B 19K or Bcl-2 were used. (In these experiments, the cells in one well received 18 ⁇ l transfection complex.) From day 10, the culture medium was replaced with fresh medium daily; This measure was based on the consideration that changing the medium would lower the concentrations of the toxins. The result of these tests is shown in FIG. 6: It showed yourself that the chosen approach is the
- Reporter plasmid pCMVßgal plus pSP65 or plus pCMV19K transfected (2 x 10 4 fibroblasts per well of a 24-well plate received 18 ⁇ l transfection complex; 500 ⁇ l complex contained either 4 ⁇ g pCMVL plus 2 ⁇ g pSP65 or 4 ⁇ g pCMVL plus 2 ⁇ g pCMV19K. 48 hours after the transfection, the cells were stained with X-gal, as described in WO 93/07283, to determine the morphology of the cells that successfully transfected the DNA
- Apoptosis trigger LPS with the anti-apoptosis gene Bcl-2 would block cell death. It was found that there was no reduction in toxicity
- A549 cells were used as control cells as well as A549 19K cells and A549-Bcl-2 cells
- control cells which were exposed to the adenovirus / pL / DNA complexes were sacrificed as a function of increasing LPS content in the sample (cf. sample 1: no LPS, with samples 2 and 3: 10 and 100 ng LPS / 6 ⁇ g DNA).
- sample 1 no LPS
- samples 2 and 3 10 and 100 ng LPS / 6 ⁇ g DNA.
- samples 2 and 3 10 and 100 ng LPS / 6 ⁇ g DNA.
- samples expressing either 19K or Bei-2 there was a decrease in toxicity to 39% (sample 2) or 77 or 60% (samples 5 or 8; 19K or Bcl-2).
- Populations of 19K or Bcl-2 expressing cells are expected to have a higher protective effect (up to 100%).
- pCMVL / pSP65 or pCMVL / pCMV19K was used in a ratio of 5: 1 each. 4 hours after the transfection, the samples were placed in fresh 10% FCS / DMEM medium or transferred to 10% FCS / DMEM medium containing 3 ⁇ M dexamethasone (Sigma). 24 hours after the transfection, the samples (double determination) were harvested and the
- Luciferase activity determined. The presence of dexamethasone in the absence of pCMV19K was shown to increase luciferase expression by a factor of 1.65. In the presence of both dexamethasone and pCMV19K, the 5,8,11-eicosatriynonic acid enhancement was 2.71-fold (Table 3).
- Adenovirus / polylysine / DNA complexes that the
- luciferase genes transfected with and without co-transfection of the E1B 19K gene, and luciferase activity was monitored over time.
- the DNA contained 5.5 ⁇ g pCMVL and 0.5 ⁇ g pSP65 (control) or pCMV19K). 2 h after the transfection, the cells were placed in standard medium (DMEM plus 10% FCS)
- Dexamethasone which indicates that the inhibition of cyclooxygenase is not sufficient to prevent the decline in gene expression. Both substances showed a synergistic effect with the EIB 19K gene in increasing gene expression.
- CELO Chicken Adenovirus CELO (Chicken Embryo Lethal Orphan) a) Isolation and cloning of CELO virus genome fragments
- the CELO virus adenovirus type 1 (ATCC VR-432) was used as starting material.
- the virus was purified after growth in chicken embryos as described by Cotten et al., 1993.
- the DNA was prepared from the virus by the proteinase K
- the entire 42.8 kb CELO virus genome obtained was digested on the one hand with EcoRI and on the other hand with HindIII.
- the mixture of fragments obtained was separated in a 0.8% agarose gel and purified on 3 mm Whatman paper and then on a Sephadex G-50 column.
- the restriction map of CELO virus is shown in the upper part of Fig. 13; it is based on the
- mapping units one
- Mapping unit is 428 base pairs). The nomenclature of the expression clones and the position of fragments 7H3 (HindIII D fragment) and 9R1 (EcoRI D fragment) are also shown. These are fragments those that showed a positive effect in the gene transfer experiments (cf. the experiments described in b).
- the fragments obtained were cloned in the expression vector pX described by Superti-Furga et al., 1991, downstream of the CMV immediate early promoter, each fragment being isolated and characterized in both orientations. b) testing of the isolated gene fragments for anti-apoptotic effects
- Example 5 Transfection experiments were carried out as indicated in Example 5, the test plasmids containing the various CELO virus fragments shown in FIG. 16.
- the amount of transfection complexes was 15 ⁇ l per 20,000 cells containing 5 ⁇ g luciferase reporter plasmid and 1 ⁇ g of the respective test plasmid (in most cases, clones were containing the fragment in both
- Expression of the control. 16 shows the relative expression (average value of 3 samples) of the test samples (5 ⁇ g luciferase reporter plasmid and 1 ⁇ g Plasmid pXCELO) against the control samples (5 ⁇ g luciferase reporter plasmid pCMVL and 1 ⁇ g empty vector) on day 14.
- a protective effect three to eightfold increase in luciferase expression could only be observed with the plasmids which contained the 7H3 and 9R1 fragments ; these two fragments originate from the same region of the CELO virus genome and have an overlapping region of approximately 80% in their sequence (see that shown in FIG. 13)
- GAM-1 is underlined, encoding, beginning with nucleotide 577, for a protein with 283 amino acids.
- the corresponding gene product was named GAM-1.
- Gene expression is not a function of the VA gene. c) Time course of gene expression in the presence of the
- transfections with the 9R1 fragment, the 7H3 fragment and the Smal / HindIII fragment of 9R1 were carried out and the luciferase expression was monitored over a longer period of time.
- BssHII cuts above the open reading frame from which the activity was suspected. Cutting with BssHII, treatment with Klenow / T4 DNA polymerase to remove the overhanging nucleotides and religion
- GAM-1 mutant # 6 L258P, L265P
- luciferase reporter plasmid was, as in the previous examples, with a luciferase reporter plasmid and either with the empty vector pSP65 (control) or with the various test plasmids (pXCELO), which each contained the fragment shown in FIG. 20 (5 ⁇ g pCMVL plus 1 ⁇ g of the respective test plasmid) transfected. 2 days after the transfection, the cells were trypsinized, counted, and a defined number of cells (20,000 per well) were (3-fold) in 24-well plates which had been coated with PolyHEMA according to Method A. 7 days after plating, the luciferase activity of the entire cell population (non-adherent plus adherent cells) was measured. Each of the values shown in Fig. 20 represents the
- Primary human fibroblasts were, as in the previous examples, with a luciferase reporter plasmid and either with the empty vector pSP65 (control) or with the various test plasmids shown in FIG. 22 (5 ⁇ g pCMVL plus 1 ⁇ g of the respective test plasmid in samples 1- 4, in samples 5 and 6 the mixture contained 0.5 .mu.g each of the
- Test plasmids transfected. 22 is the
- Luciferase expression was shown 22 days after transfection, with each value averaging 3
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US08/750,180 US6284880B1 (en) | 1994-05-30 | 1995-05-26 | Chicken embryo lethal orphan (CELO) virus GAM-1 |
AU26160/95A AU709148B2 (en) | 1994-05-30 | 1995-05-26 | Method for introducing foreign matter into higher eukaryotic cells |
EP95920887A EP0767840A2 (de) | 1994-05-30 | 1995-05-26 | Verfahren zum einbringen von fremdmaterial in höhere eukaryotische zellen |
JP8500282A JPH10500578A (ja) | 1994-05-30 | 1995-05-26 | 外来物質を高等真核生物細胞に導入する方法 |
MX9605941A MX9605941A (es) | 1994-05-30 | 1995-05-26 | Procedimiento para introducir material extraño en celulas eucarioticas superiores. |
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DE4418825A DE4418825A1 (de) | 1994-05-30 | 1994-05-30 | Verfahren zum Einbringen von Fremdmaterial in höhere eukaryotische Zellen |
DEP4418825.0 | 1994-05-30 | ||
DEP4442587.2 | 1994-11-30 | ||
DE19944442587 DE4442587A1 (de) | 1994-11-30 | 1994-11-30 | Verfahren zum Einbringen von Fremdmaterial in höhere eukaryotische Zellen |
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WO1999015677A1 (en) * | 1997-09-19 | 1999-04-01 | The Trustees Of The University Of Pennsylvania | Method for gene transfer using bcl2 and compositions useful therein |
WO1999041398A1 (en) * | 1998-02-11 | 1999-08-19 | Genvec, Inc. | Vectors, cells and methods for the production of deleterious viral eukaryotic gene transfer vectors |
US6335016B1 (en) | 1996-04-20 | 2002-01-01 | Boehringer Ingelheim International Gmbh | Chicken embryo lethal orphan (CELO) virus |
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GB9908195D0 (en) | 1999-04-09 | 1999-06-02 | Microbiological Res Authority | Treatment of intracellular infection |
US20050013870A1 (en) * | 2003-07-17 | 2005-01-20 | Toby Freyman | Decellularized extracellular matrix of conditioned body tissues and uses thereof |
US7033673B2 (en) * | 2003-07-25 | 2006-04-25 | Analytical Services & Materials, Inc. | Erosion-resistant silicone coatings for protection of fluid-handling parts |
US20070178137A1 (en) * | 2006-02-01 | 2007-08-02 | Toby Freyman | Local control of inflammation |
JP2010536345A (ja) * | 2007-08-14 | 2010-12-02 | グラクソスミスクライン・リミテッド・ライアビリティ・カンパニー | 新規な方法および細胞系 |
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WO1993007283A1 (de) * | 1991-09-30 | 1993-04-15 | Boehringer Ingelheim International Gmbh | Zusammensetzung für das einbringen von nukleinsäure-komplexen in höhere eukaryotische zellen |
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US5354844A (en) | 1989-03-16 | 1994-10-11 | Boehringer Ingelheim International Gmbh | Protein-polycation conjugates |
US5521291A (en) | 1991-09-30 | 1996-05-28 | Boehringer Ingelheim International, Gmbh | Conjugates for introducing nucleic acid into higher eucaryotic cells |
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Non-Patent Citations (7)
Title |
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ANNALS NEW YORK ACADEMY OF SCIENCES, Bd. 660, 1992, Seiten 136-153, XP002003027 ZATLOUKAL, K. ET AL.: "Transferrinfection: A Highly Efficient Way to Express Gene Constructs in Eukaryotic Cells" * |
CANCER RESEARCH, Bd. 52, Oktober 1992, Seiten 5407-5411, XP002003025 MIYASHITA, T. UND REED, J.C.: "bcl-2 Gene Transfer Increases Relative Resistance of S49.1 and WEHI7.2 Lymphoid Cells to Cell Death and DNA Fragmentation Induced by Glucocorticoids and Multiple Chemotherapeutic Drugs" * |
EMBL Database ID-Nr.: AAORFSA Accession-Nr.: Z22864 XP002003029 * |
GENES & DEVELOPMENT, Bd. 7, 1993, Seiten 546-554, XP002003024 DEBBAS, M. UND WHITE, E.: "Wild-type p53 mediates apoptosis by E1A, which is inhibited by E1B" * |
HUMAN GENE THERAPY, Bd. 3, 1992, Seiten 147-154, XP002003028 CURIEL, D.T. ET AL.: "High-Efficiency Gene Transfer Mediated by Adenovirus Coupled to DNA-Polylysine Complexes" * |
J.VIROLOGY, Bd. 68, Nr. 10, Oktober 1994, Seiten 6553-6566, XP002003026 CHIOU, S.-K. ET AL.: "Functional Complementation of the Adenovirus E1B 19-Kilodalton Protein with Bcl-2 in the Inhibition of Apoptosis in Infected Cells" * |
PROC.NATL.ACAD.SCI.USA, Bd. 89, August 1992, Seiten 7742-7746, XP002003023 RAO, L. ET AL.: "The adenovirus E1A proteins induce apoptosis, which is inhibited by the E1B 19-kDa and Bcl-2 proteins" * |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6335016B1 (en) | 1996-04-20 | 2002-01-01 | Boehringer Ingelheim International Gmbh | Chicken embryo lethal orphan (CELO) virus |
US6773709B2 (en) | 1996-04-20 | 2004-08-10 | Boehringer Ingelheim International Gmbh | Chicken embryo lethal (CELO) virus |
WO1999015677A1 (en) * | 1997-09-19 | 1999-04-01 | The Trustees Of The University Of Pennsylvania | Method for gene transfer using bcl2 and compositions useful therein |
WO1999041398A1 (en) * | 1998-02-11 | 1999-08-19 | Genvec, Inc. | Vectors, cells and methods for the production of deleterious viral eukaryotic gene transfer vectors |
AU753027B2 (en) * | 1998-02-11 | 2002-10-03 | Genvec, Inc. | Vectors, cells and methods for the production of deleterious viral eukaryotic gene transfer vectors |
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WO1995033062A3 (de) | 1996-07-18 |
US6284880B1 (en) | 2001-09-04 |
MX9605941A (es) | 1997-12-31 |
AU709148B2 (en) | 1999-08-19 |
EP0767840A2 (de) | 1997-04-16 |
CA2190963A1 (en) | 1995-12-07 |
CO4410254A1 (es) | 1997-01-09 |
JPH10500578A (ja) | 1998-01-20 |
AU2616095A (en) | 1995-12-21 |
US20020049313A1 (en) | 2002-04-25 |
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