Host-vector system which can be used in gene therapy

Abstract

The invention relates to a system for expressing a transgene in a target cell or a human or animal cell, characterized in that it consists of a eukaryotic cell established as a line, into which there have been transfected: a) a recombinant viral sequence in which a gene has been deleted totally or partially and substituted by the said transgene at the level of this gene; b) a nucleic acid sequence including a sequence encoding the deleted protein, which sequence is in dependence on a promoter and is combined, where appropriate, with the said transgene, and flanked at its 3′ end a polyadenylation site; the said recombinant viral genome and the said sequence, carried by one or two plasmid supports, being capable of trans-complementing each other and allowing the said host cell to produce defective infectious viruses.

1 . Host-vector system which makes it possible to express a transgene in a target cell or a human or animal tissue, characterized in that it consists of a eukaryotic cell established as a line, into which there have been transfected: 
 a) a recombinant pseudo-retroviral sequence in which the env gene has been deleted totally or partially and substituted by the said transgene at the level of the env gene as represented in FIG. 1 a;      b) a nucleic acid sequence including a sequence encoding an envelope protein, which sequence is in dependence on a promoter and is combined, where appropriate, with the said transgene, and flanked at its 3′ end a polyadenylation site and represented in FIG. 1 b;      the said recombinant viral genome and the said sequence being capable of trans-complementing each other and allowing the said host cell to produce defective infectious viruses lacking the env gene.    
     
     
         2 . System according to  claim 1 , characterized in that the sequence in a) and the sequence in b) are carried by two separate plasmids.  
     
     
         3 . System according to  claim 1 , characterized in that the recombinant sequences in a) and in b) are carried by the same plasmid provided that the sequence  1   b ) is outside the region for regulating the expression of the recombinant pseudo-retroviral sequence in a), in particular outside the LTRs.  
     
     
         4 . System according to  claim 3 , characterized in that the plasmid contains, in addition, one or two AAV ITR sequences situated upstream, or upstream and downstream of the LTRs.  
     
     
         5 . System according to one of  claims 1  to  4 , characterized in that the nucleic acid sequence including a sequence encoding an envelope protein comprises, in addition, upstream of the said sequence, a homologous sequence of the defective recombinant retrovirus used in the host-vector system, chosen especially from all or part of the gag gene or all or part of the pol gene.  
     
     
         6 . System according to one of  claims 1  to  5 , characterized in that the transgene to be expressed is a suicide gene, especially the Herpes Simplex virus thymidine kinase (HSV1-TK).  
     
     
         7 . System according to one of  claims 1  to  5 , characterized in that the recombinant pseudoviral sequence is derived from the Moloney genome MuLV, the LTR sequences in 5′ or in 3′ being of wild-type, mutant or combined origin.  
     
     
         8 . System according to one of  claims 1  to  5 , characterized in that the nucleic acid sequence encoding the env gene is of viral origin and is chosen especially from the sequences encoding the MuLV, VSV, EIV and rabies env gene.  
     
     
         9 . System according to one of  claims 1  to  5 , characterized in that the nucleic acid sequence encoding the env gene is of cellular origin and consists of a sequence encoding a membrane protein, allowing the targeting of the viral particle on a specific ligand, especially a protein encoding a CD4-type receptor.  
     
     
         10 . System according to one of  claims 1  to  5 , characterized in that the env gene is a chimeric protein in which the carboxy-terminal end is derived from intramembrane sequences of the Moloney envelope.  
     
     
         11 . System according to one of  claims 1  to  10 , characterized in that the sequence containing the env gene may be introduced by infection with a viral vector, especially an adenovirus.  
     
     
         12 . System according to one of  claims 1  to  10 , characterized in that the recombinant pseudo-viral sequence and the sequence carrying the env. gene are enveloped in vehicles such as liposomes, and introduced into the cells by transfection.  
     
     
         13 . System according to  claim 12 , characterized in that the transfectable cells are either host cells, or target cells.  
     
     
         14 . System according to one of  claims 1  to  10 , characterized in that the eukaryotic cell is a cell established as a line and may be chosen especially from the fibroblast lines such as the 3T3 line, or lines capable of being cultured in suspension, such as mouse myeloma cells, or VERO cells.  
     
     
         15 . System according to one of  claims 1  to  10 , characterized in that the cells used are Lepidoptera cells.  
     
     
         16 . Process for expressing a transgene for gene therapy which consists in using the following steps: 
 a) construction of a host-vector system obtained by transfection, into a eukaryotic host cell, on the one hand, of a pseudo-retroviral sequence in which the env gene is deleted totally and replaced with the transgene, especially at the level of the ATG of the said env gene and, on the other hand, a nucleic acid sequence containing, in its structure, a sequence encoding an envelope protein under the control of a promoter, where appropriate combined with the transgene and flanked at its 3′ end by a polyadenylation sequence,    b) bringing the said system into contact with the cells in which the tranagene has to be expressed,    c) where appropriate, again transferring, into the target cells, the abovementioned sequence containing the env gene.    
     
     
         17 . Process according to  claim 16 , characterized in that the pseudo-retroviral sequences and the sequence carrying an env. gene are enveloped in vehicles.  
     
     
         18 . Process according to  claim 17 , characterized in that the transfection vehicle is chosen by liposomes, cationic lipids, polylysine derivatives, inactivated adenoviruses or a ballistic means.  
     
     
         19 . Process according to  claim 16  or  17 , characterized in that the pseudo-retroviral sequence and the sequence carrying the env. gene are carried by the same plasmid.  
     
     
         20 . Process according to  claim 19 , characterized in that the plasmid contains, in addition, one or two AAV ITR sequences situated upsteam, or upstream and downstream of the LTRe.  
     
     
         21 . Process according to one of  claims 16  to  19 , characterized in that the transgene is a suicide gene, especially the HSV1 thymidine kinase gene, and in that the cells into which the said transgene is integrated are destroyed by addition of a nucleotide analog, especially gancyclovir or acyclovir.  
     
     
         22 . Process according to  claim 16 , characterized in that the pseudo-retroviral sequence is directly derived from the Moloney virus MuLV, the LTR sequences in 5′ or in 3′ being of wild-type, mutant or combined origin.  
     
     
         23 . Process according to one of  claims 16  to  19 , characterized in that the sequence encoding the env gene is chosen especially from the sequences of viruses encoding the said gene, especially MULV, or VSV, RIV, or the rabies virus or sequences encoding cell membrane proteins, especially CD4.  
     
     
         24 . Process according to one of  claims 16  to  19 , characterized in that the sequence containing the env gene is introduced into the target cell by a viral vector, especially derived from an adenovirus.  
     
     
         25 . Process according to one of  claims 16  to  19 , characterized in that the sequence containing the env gene is introduced into the target cells by physical methods, especially by bombardment, fusion of liposomes, microinjection.  
     
     
         26 . Process according to one of  claims 16  to  19 , characterized in that the cell used is chosen from established lines not expressing the transgene of interest considered, especially mouse 3T3 fibroblast lines, mouse myelomas or insect cells, or VERO cells.  
     
     
         27 . Process allowing the expression of a transgene for gene therapy which consists in a double transfection of the target cells, on the one hand, with defective infectious viruses obtained by the host-vector system of  claim 1  and, on the other hand, with a nucleic acid sequence containing in its structure a retrovirus envelope gene, the in vivo co-expression in trans of the two sequences allowing the expression of infectious recombinant viral particles, but lacking the said env gene in their genome.  
     
     
         28 . Use, in the manufacture of a medicinal product for gene therapy, of a host-vector system obtained by the following successive steps: 
 a) simultaneous transfection, into a eukaryotic host cell, on the one hand, of a pseudo-retroviral sequence in which the env gene is deleted totally and replaced with the transgene and, on the other hand, a nucleic acid sequence containing, in its structure, a sequence encoding an envelope protein under the control of a promoter, where appropriate, combined with the transgene and flanked at its 3′ end by a polyadenylation sequence, and, where appropriate, combined at its 3′ end with a homologous sequence of the defective recombinant retrovirus used in the host-vector system of a gene encoding a retroviral protein,    b) bringing the said system into contact with the cells in which the transgene has to be expressed,    c) where appropriate, again transferring, into the target cells, the abovementioned sequence containing the env gene.    
     
     
         29 . Use according to  claim 28 , characterized in that the pseudo-retroviral sequence and the nucleic acid sequence carrying an env. gene are carried by a single plasmid.  
     
     
         30 . Use according to  claim 29 , characterized in that the plasmid contains, in addition, one or two AAV ITR sequences situated upstream, or upstream and downstream of the LTRs.  
     
     
         31 . Use according to  claim 28 , characterized in that the pseudo-retroviral sequence and the sequence carrying the env. gene are carried by two separate plasmids.  
     
     
         32 . Use according to one of  claims 27  to  30 , characterized in that the gene to be expressed is a suicide gene.  
     
     
         33 . Use according to  claim 32 , characterized in that the suicide gene is the thymidine kinase gene, the target cells in which the gene is expressed being destroyed by addition of a nucleoside analog, especially acyclovir or gancyclovir.  
     
     
         34 . Medicinal product which can be used in gene therapy, characterized in that it contains, as active ingredient, vehicles containing a recombinant retroviral sequence substituted, at the level of the env. gene, by a transgene of interest, and outside the region for regulating the expression of the retroviral sequences, especially outside the LTRs, a sequence carrying an env. gene in dependence on a promoter.  
     
     
         35 . Medicinal product according to  claim 34 , characterized in that the plasmid contains, in addition, one or two AAV ITR sequences situated upstream, or upstream and downstream of the LTRs.  
     
     
         36 . Medicinal product which can be used in gene therapy, characterized in that it contains, as active ingredient, eukaryotic cells which have been subjected to a double transfection and represented by the host-vector system according to  claim 1 .  
     
     
         37 . Recombinant viral vector containing a transgene of interest, which can be used in gene therapy, characterized in that: 
 a gene essential for the constitution of infectious viral particles is substituted by a transgene of interest,    this essential gene is present on the same vector, or a different vector, but is no longer in dependence on the viral promoters and,    the product of this gene acts in trans and makes it possible to reconstitute defective viral particles.    
     
     
         38 . Vector according to  claim 37 , characterized in that the virus is a retrovirus, and the transgene is substituted in the env gene.  
     
     
         39 . Vector according to  claim 37 , characterized in that the virus is an adenovirus and the transgene is substituted in the E1A gene.  
     
     
         40 . Use of a vector according to one of  claims 37  to  39 , in the manufacture of a medicinal product which can be used in gene therapy.