Method for introducing foreign material into higher eukaryotic cells

Abstract

The toxicity problems which occur when foreign material is introduced into higher eukaryotic cells, particularly during transfection with DNA, are countered by causing gene products to be expressed in the cell, which block the apoptosis triggered by the transfection process, and/or by treating the cells with anti-inflammatory substances. Preferably, Bcl-2, E1B 19K or an anti-apoptotically active gene of chicken adenovirus CELO is used as the anti-apoptosis gene whilst the inflammatory substance used is adenovirus VA1, which is introduced into the cell in the form of VA1-DNA. Using these methods, long-lasting gene expression can be achieved.

1 . Process for treating higher eukaryotic cells, particularly for introducing foreign material such as nucleic acid into the cells, characterised in that, apart from the foreign material and the transfection components 
 a) one or more nucleic acid molecules, particularly DNA molecules, are introduced into the cell, the expression products of which at least partially block the apoptosis of the host cell initiated by the introduction of the foreign material, and/or    b) the inflammatory response of the cells is at least partially inhibited by    i) treating the cells externally with one or more anti-inflammatory substances and/or    ii) one or more anti-inflammatory substances or the nucleic acid molecules, particularly DNA molecules, coding therefor are introduced into the cells.    
     
     
         2 . Process according to  claim 1 , characterised in that the foreign material is DNA which is complexed with a polycation optionally conjugated with a ligand for the target cell, and in that the DNA complex is introduced into the cells in the presence of adenovirus or adenovirus/polycation conjugate.  
     
     
         3 . Process according to  claim 1  or  2 , characterised in that the DNA molecule defined in a) having an anti-apoptosis activity codes for human Bcl-2.  
     
     
         4 . Process according to  claim 1  or  2 , characterised in that the DNA molecule defined in a) having an anti-apoptosis activity codes for adenovirus E1B 19K.  
     
     
         5 . Process according to  claim 1  or  2 , characterised in that the DNA molecule with an anti-apoptosis activity codes for a gene product which inactivates p53.  
     
     
         6 . Process according to  claim 1  or  2 , characterised in that the DNA molecule defined in a) is a SmaI/HindIII fragment of the CELO virus genome which is located in the region between about 84.3 and 88.7 mapping units, or a DNA sequence which contains this fragment.  
     
     
         7 . Process according to  claim 6 , characterised in that the DNA molecule has the nucleotide sequence shown in SEQ ID NO:1 or a partial sequence thereof.  
     
     
         8 . Process according to  claim 6 , characterised in that the DNA molecule is an EcoRI fragment of the CELO virus genome which is located in the region between the mapping units 81.2 and 92.7.  
     
     
         9 . Process according to  claim 6 , characterised in that the DNA molecule is a HindIII fragment located in the region between the mapping units 77.8 and 88.7.  
     
     
         10 . Process according to  claim 6 , characterised in that the DNA molecule has the open reading frame contained in the sequence according to SEQ ID NO:1 or codes for a gene product which is a functional derivative of the gene product defined by this reading frame.  
     
     
         11 . Process according to one of  claims 2  to  10 , characterised in that the DNA molecule with anti-apoptosis activity is present, together with the DNA to be introduced into the cell, as component of a DNA-polycation complex.  
     
     
         12 . Process according to  claim 11 , characterised in that the DNA to be introduced into the cell and the DNA molecule with an anti-apoptosis activity are present on separate plasmids.  
     
     
         13 . Process according to  claim 11 , characterised in that the DNA to be introduced into the cell and the DNA molecule with an anti-apoptosis activity are both present on a plasmid.  
     
     
         14 . Process according to  claim 1 , characterised in that the anti-inflammatory substance is adenovirus VA1.  
     
     
         15 . Process according to  claim 1  or  14 , characterised in that the anti-inflammatory substance is introduced into the cell in the form of the DNA coding for it.  
     
     
         16 . Process according to  claim 2 ,  14  or  15 , characterised in that the VA1-DNA is present, together with the DNA to be introduced into the cell, as a component of a DNA-polycation complex.  
     
     
         17 . Process according to  claim 16 , characterised in that the complex also contains the DNA molecule defined in a).  
     
     
         18 . Process according to  claim 16  or  17 , characterised in that the two or three DNA molecules are present on separate plasmids.  
     
     
         19 . Process according to  claim 16  or  17 , characterised in that the two or three DNA molecules are present together on one plasmid.  
     
     
         20 . Process according to  claim 1 , characterised in that a DNA molecule having an anti-apoptosis activity is introduced into the cell and the cells are treated externally with an anti-inflammatory substance.  
     
     
         21 . Process according to  claim 20 , characterised in that the anti-inflammatory substance inhibits the activation of phospholipase A2.  
     
     
         22 . Process according to  claim 15 , characterised in that the anti-inflammatory substance is a glucocorticoid.  
     
     
         23 . Process according to  claim 22 , characterised in that the glucocorticoid is dexamethasone.  
     
     
         24 . Process according to  claim 20 , characterised in that the anti-inflammatory substance inhibits a metabolite of arachidonic acid.  
     
     
         25 . Process according to  claim 24 , characterised in that the anti-inflammatory substance is ibuprofen.  
     
     
         26 . Process according to  claim 24 , characterised in that the anti-inflammatory substance is acetylsalicylic acid.  
     
     
         27 . Process according to  claim 24 , characterised in that the anti-inflammatory substance is nordihydroguairetic acid (NDGA).  
     
     
         28 . Process according to  claim 24 , characterised in that the anti-inflammatory substance is 5,8,11-eicosatriynoic acid.  
     
     
         29 . Process according to  claim 24 , characterised in that the anti-inflammatory substance is indomethacin.  
     
     
         30 . DNA molecule containing the nucleotide sequence shown in SEQ ID NO:1 or a partial sequence thereof coding for a functional gene product having an anti-apoptotic effect.  
     
     
         31 . DNA molecule according to  claim 30 , characterised in that it has the open reading frame contained in the sequence according to SEQ ID NO:1, including degenerate variants, or mutants, which code for a functional gene product having an anti-apoptotic activity.  
     
     
         32 . Polypeptide, characterised in that it has the amino acid sequence shown in SEQ ID NO:2.  
     
     
         33 . Transfection complex containing a nucleic acid molecule which is to be expressed in the cell, which is complexed with a polycation optionally conjugated with a ligand for the target cell, as well as adenovirus or an adenovirus polycation conjugate, characterised in that it also contains a DNA molecule having an anti-apoptosis activity and/or a DNA molecule coding for a substance having an anti-inflammatory effect.  
     
     
         34 . Pharmaceutical preparation containing a transfection complex according to  claim 33 , in which the nucleic acid molecule to be expressed in the cell is a therapeutically or gene-therapeutically active DNA molecule.