Circular DNA expression cassettes for gene transfer

Abstract

Double-stranded DNA molecules characterised in that they are circular and in that they essentially include one or more genes of interest.

1 . Double-stranded DNA molecule, characterized in that: 
 it is in circular and supercoiled form,    it comprises an expression cassette consisting of a gene of interest under the control of a transcription promoter and a transcription terminator which are active in mammalian cells,    it lacks an origin of replication,    it lacks a marker gene, and    it comprises a region resulting from the site-specific recombination between two sequences, said region being located outside the expression cassette.    
     
     
         2 . Molecule according to  claim 1 , characterized in that it contains, in addition, a sequence capable of interacting specifically with a ligand.  
     
     
         3 . Molecule according to  claim 2 , characterized in that the sequence capable of interacting specifically with a ligand is a sequence capable of forming a triple helix by hybridization with a specific oligonucleotide.  
     
     
         4 . Molecule according to  claim 3 , characterized in that the sequence capable of forming a triple helix comprises from 5 to 30 base pairs.  
     
     
         5 . Molecule according to  claim 3  or  4 , characterized in that the sequence capable of forming a triple helix is a homopurine-homopyrimidine sequence.  
     
     
         6 . Molecule according to  claim 1 , characterized in that it comprises a sequence resulting from the site-specific recombination between two att attachment sequences or between two recognition sequences of the resolvase of a transposon or parA of RK2.  
     
     
         7 . Molecule according to one of the preceding claims, characterized in that it comprises, in addition, an mrs sequence originating from the par locus of RK2, permitting the resolution of multimers.  
     
     
         8 . Molecule according to one of the preceding claims, characterized in that the gene of interest is a nucleic acid coding for a therapeutic, vaccinal, agricultural or veterinary product.  
     
     
         9 . Molecule according to one of the preceding claims, characterized in that it is obtained by excision from a plasmid or chromosome, by site-specific recombination.  
     
     
         10 . Recombinant DNA, comprising an expression cassette consisting of a gene of interest under the control of a transcription promoter and a transcription terminator which are active in mammalian cells, flanked by two sequences permitting a site-specific recombination, positioned in the direct orientation.  
     
     
         11 . Recombinant DNA according to  claim 13 , characterized in that it is a replicative plasmid comprising: 
 a) an origin of replication and optionally a marker gene,    b) two sequences permitting a site-specific recombination, positioned in the direct orientation, and,    c) placed between said sequences b), an expression cassette consisting of a gene of interest under the control of a transcription promoter and a transcription terminator which are active in mammalian cells.    
     
     
         12 . Recombinant DNA according to claims  10  and  11 , characterized in that the sequences permitting site-specific recombination are sequences capable of recombining specifically in the presence of a recombinase.  
     
     
         13 . Recombinant DNA according to  claim 12 , characterized in that the recombinase is chosen from the recombinases of the integrase family of phage lambda and of the resolvase family of the transposon Tn3.  
     
     
         14 . Recombinant DNA according to one of  claims 10  to  13 , characterized in that the sequences permitting site-specific recombination are derived from a bacteriophage.  
     
     
         15 . Recombinant DNA according to  claim 14 , characterized in that the sequences permitting site-specific recombination consist of the att attachment sequences of a bacteriophage, or derived sequences.  
     
     
         16 . Recombinant DNA according to  claim 15 , characterized in that the sequences permitting site-specific recombination consist of the attachment sequences of bacteriophage lambda, P22, Φ80, P1 or HP1 or alternatively of plasmid pSAM2 or the 2 plasmid, or derived sequences.  
     
     
         17 . Recombinant DNA according to  claim 16 , characterized in that the sequences permitting site-specific recombination comprise all or part of the sequences SEQ ID No.1, 2, 6, 7, 8, 9, 10, 11, 12, 13 or 14.  
     
     
         18 . Plasmid, characterized in that it comprises: 
 (a) a bacterial origin of replication and optionally a marker gene;    (b) the attP and attB sequences of a bacteriophage selected from the phages lambda, P22, Φ80, P1 and HP1 or of plasmid pSAM2 or the 2 plasmid, positioned in the direct orientation; and,    (c) placed between said sequences, an expression cassette consisting of a gene of interest under the control of a transcription promoter and a transcription terminator which are active in mammalian cells.    
     
     
         19 . Plasmid according to  claim 18 , characterized in that the sequences permitting site-specific recombination consist of the attachment sequences of bacteriophage lambda.  
     
     
         20 . Recombinant DNA according to  claim 14 , characterized in that the sequences permitting site-specific recombination are derived from bacteriophage P1.  
     
     
         21 . Plasmid, characterized in that it comprises: 
 (a) a bacterial origin of replication and optionally a marker gene;    (b) the inverted repeat sequences of bacteriophage P1 (loxP region), positioned in the direct orientation; and,    (c) placed between said sequences (b), an expression cassette consisting of a gene of interest under the control of a transcription promoter and a transcription terminator which are active in mammalian cells.    
     
     
         22 . Recombinant DNA according to one of  claims 10  to  13 , characterized in that the sequences permitting site-specific recombination are derived from a transposon.  
     
     
         23 . Recombinant DNA according to  claim 22 , characterized in that the sequences permitting site-specific recombination consist of the recognition sequences of the resolvase of the transposon Tn3, Tn21 or Tn522, or derived sequences.  
     
     
         24 . Recombinant DNA according to  claim 23 , characterized in that the sequences permitting site-specific recombination comprise all or part of the sequence SEQ ID No.15.  
     
     
         25 . Recombinant DNA according to one of  claims 10  to  13 , characterized in that the sequences permitting site-specific recombination are derived from the par region of plasmid RP4.  
     
     
         26 . Recombinant DNA according to one of  claims 10  to  25 , characterized in that it comprises, in addition, a sequence capable of interacting specifically with a ligand.  
     
     
         27 . Plasmid, characterized in that it comprises: 
 a) an origin of replication and optionally a marker gene,    b) two sequences permitting a site-specific recombination, positioned in the direct orientation, and,    c) placed between said sequences b), one or more genes of interest and a sequence capable of interacting specifically with a ligand.    
     
     
         28 . Plasmid, characterized in that it comprises: 
 a) an origin of replication and optionally a marker gene,    b) two sequences permitting a site-specific recombination, positioned in the direct orientation, and,    c) placed between said sequences b), one or more genes of interest and an mrs sequence originating from the par locus of RK2, permitting the resolution of multimers.    
     
     
         29 . Plasmid, characterized in that it comprises: 
 a) an origin of replication and optionally a marker gene,    b) two sequences permitting a site-specific recombination, positioned in the direct orientation, and,    c) placed between said sequences b), one or more genes of interest, an mrs sequence originating from the par locus of RK2, permitting the resolution of multimers, and a sequence capable of interacting specifically with a ligand.    
     
     
         30 . Plasmid, characterized in that it comprises: 
 a) an origin of replication and optionally a marker gene,    b) two sequences permitting an integrase-dependent site-specific recombination, positioned in the direct orientation, and two sequences permitting a resolvase-dependent site-specific recombination, positioned next to the first two sequences and also in the direct orientation, and,    c) placed between said sequences b), one or more genes of interest and optionally a sequence capable of interacting specifically with a ligand.    
     
     
         31 . Plasmid according to claims  27 ,  29  and  30 , characterized in that the sequence capable of interacting specifically with a ligand is defined as in  claims 2  to  5 .  
     
     
         32 . Recombinant cell containing a plasmid according to  claim 14 ,  27 ,  29  or  30 .  
     
     
         33 . Recombinant cell comprising, inserted into its genome, one or more copies of a recombinant DNA according to  claim 10 .  
     
     
         34 . Recombinant cell according to  claim 32  or  33 , characterized in that it is a bacterium.  
     
     
         35 . Recombinant cell according to  claim 32  or  33 , characterized in that it is a eukaryotic cell.  
     
     
         36 . Recombinant cell according to  claim 34 , characterized in that it is the bacterium  E. coli  D1210HP.  
     
     
         37 . Pharmaceutical composition comprising at least one DNA molecule according to one of  claims 1  to  9 .  
     
     
         38 . Method for the preparation of a DNA molecule according to one of  claims 1  to  19 , characterized in that a culture of host cells containing a recombinant DNA as defined in  claim 10  is brought into contact with the recombinase enabling site-specific recombination to be induced in vivo.  
     
     
         39 . Method according to  claim 38 , characterized in that the culture of host cells is a culture of cells according to  claim 32 .  
     
     
         40 . Method according to  claim 38 , characterized in that the culture of host cells is a culture of cells according to  claim 33 .  
     
     
         41 . Method according to one of  claims 38  to  40 , characterized in that the culture is brought into contact with the recombinase by transfection or infection of the cell culture with a plasmid or a phage containing the gene for said recombinase.  
     
     
         42 . Method according to one of  claims 38  to  40 , characterized in that the culture is brought into contact with the recombinase by induction of the expression of a gene coding for said recombinase, present in the host cell.  
     
     
         43 . Method according to  claim 42 , characterized in that the host cell contains, integrated in its genome, the recombinase gene having a temperature-regulated expression, and the culture is brought into contact with the recombinase by culturing at the induction temperature.  
     
     
         44 . Method according to  claim 43 , characterized in that the host cell used contains a lysogenic phage integrated in its genome, containing the gene for said recombinase.  
     
     
         45 . Method for the preparation of a DNA molecule according to one of  claims 1  to  9 , characterized in that a plasmid preparation according to  claim 11  is brought into contact with the recombinase enabling site-specific recombination to be induced in vitro.  
     
     
         46 . Method according to  claim 37  or  45 , characterized in that it comprises an additional step of purification of the minicircle.  
     
     
         47 . Method according to  claim 46 , characterized in that the purification comprises a step of bringing a solution containing the minicircle into contact with a specific ligand, optionally grafted onto a support.  
     
     
         48 . Method according to  claim 47 , characterized in that the solution containing the minicircle is brought into contact with an oligonucleotide, optionally grafted onto a support, capable of forming, by hybridization, a triple helix with a specific sequence present in the minicircle.