Circular dna molecule having a conditional origin of replication, process for their preparation and their use in gene therapy

Abstract

A circular DNA molecule, useful for gene therapy, comprising at least one nucleic acid sequence of interest, characterised in that the region allowing the replication thereof has an origin of replication with a functionality in a host cell that requires the presence of at least one specific protein foreign to said host cell. A method for preparing same, cells incorporating said DNA molecules and uses thereof in gene therapy are also described.

1 - 18 . (canceled) 
     
     
         19 . A prokaryotic recombinant host cell comprising (a) a heterologous replication initiation protein that activates a conditional origin of replication, wherein the replication initiation protein is encoded by gene pir 16; and (b) a gene encoding a protein that is essential for host cell viability under defined culture conditions, wherein the gene has a codon that is recognized by a suppressor tRNA. 
     
     
         20 . The prokaryotic recombinant host cell according to  claim 19 , wherein the codon that is recognized by the suppressor tRNA is an amber codon. 
     
     
         21 . The prokaryotic recombinant host cell according to  claim 19 , wherein the gene having the codon that is recognized by the suppressor tRNA is a gene involved in the biosynthesis of a metabolite or a catabolism gene. 
     
     
         22 . The prokaryotic recombinant host cell according to  claim 21 , wherein the metabolite is an amino acid. 
     
     
         23 . The prokaryotic recombinant host cell according to  claim 22 , wherein the amino acid is arginine. 
     
     
         24 . The prokaryotic recombinant host cell according to  claim 21 , wherein the gene having the codon that is recognized by the suppressor tRNA is the argE gene. 
     
     
         25 . The prokaryotic recombinant host cell according to  claim 19 , further comprising an extrachromosomal DNA molecule, wherein the extrachromosomal DNA molecule comprises a gene coding for a suppressor tRNA. 
     
     
         26 . The prokaryotic recombinant host cell according to  claim 25 , wherein the suppressor tRNA is for an amber codon. 
     
     
         27 . The prokaryotic recombinant host cell according to  claim 19 , wherein the conditional origin of replication is from bacterial plasmid R6K. 
     
     
         28 . A method of expressing a gene of interest in an animal cell comprising in vitro transfection of the animal cell with an extrachromosomal DNA molecule comprising:
 (a) a gene of interest operably linked to a promoter and a polyadenylation site;   (b) a conditional origin of replication whose functionality in a prokaryotic host cell requires a replication initiating protein that is specific for the origin of replication, foreign to the prokaryotic host cell, and not encoded by the extrachromosomal DNA;   (c) a gene encoding a suppressor tRNA; and   (d) a target for a site-specific recombinase.   
     
     
         29 . The method of  claim 28 , wherein the conditional origin of replication is the γ origin of replication of the plasmid R6K. 
     
     
         30 . The method of  claim 28 , wherein the gene encoding the suppressor tRNA is for amber codons. 
     
     
         31 . The method of  claim 28 , wherein the target for a site-specific recombinase is a cer fragment from ColE1. 
     
     
         32 . The method of  claim 28 , wherein the animal cell is a human cell. 
     
     
         33 . The method of  claim 28 , wherein the gene of interest encodes a therapeutic protein. 
     
     
         34 . The method of  claim 33 , wherein the method further comprises administering the transfected animal cells to an animal. 
     
     
         35 . A method of expressing a gene of interest in an animal cell comprising injecting into an animal a composition comprising an extrachromosomal DNA molecule comprising:
 (a) a gene of interest operably linked to a promoter and a polyadenylation site;   (b) a conditional origin of replication whose functionality in a prokaryotic host cell requires a replication initiating protein that is specific for the origin of replication, foreign to the prokaryotic host cell, and not encoded by the extrachromosomal DNA;   (c) a gene encoding a suppressor tRNA; and   (d) a target for a site-specific recombinase.   
     
     
         36 . The method of  claim 35 , wherein the composition comprises a cationic polymer or lipid. 
     
     
         37 . The method of  claim 35 , wherein the conditional origin of replication is the γ origin of replication of the plasmid R6K. 
     
     
         38 . The method of  claim 35 , wherein the gene encoding the suppressor tRNA is for amber codons. 
     
     
         39 . The method of  claim 35 , wherein the target for a site-specific recombinase is a cer fragment from ColE1. 
     
     
         40 . The method of  claim 35 , wherein the animal cell is a human cell. 
     
     
         41 . The method of  claim 35 , wherein the gene of interest encodes a therapeutic protein.