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

Abstract

A prokaryotic recombinant host cell comprising a heterologous replication initiation protein that activates a conditional origin of replication and an extrachromosomal DNA molecule comprising a heterologous therapeutic gene and a conditional origin of replication whose functionality in the prokaryotic recombinant host cell requires a replication initiating protein which is foreign to the host cell is described. The host cell may comprise a pir gene having at least one mutation, which may occur in the pir gene copy number control region, the pir gene leucine zipper-like motif, or the pir gene DNA binding region.

1 - 49 . (canceled) 
     
     
         50 . A plasmid comprising a heterologous pir gene having at least one mutation in the pir gene leucine zipper-like motif or the pir gene DNA binding region. 
     
     
         51 . The plasmid of  claim 50 , wherein the heterologous pir gene comprises at least one mutation in the pir gene leucine zipper-like motif. 
     
     
         52 . The plasmid of  claim 51 , further comprising a mutation in the pir gene DNA binding region. 
     
     
         53 . The plasmid of  claim 50 , wherein the heterologous pir gene comprises at least one mutation in the DNA binding region and the leucine zipper-like motif. 
     
     
         54 . The plasmid of  claim 50 , wherein the mutation in the DNA binding region is the 114C mutation. 
     
     
         55 . The plasmid of  claim 50 , wherein the mutation in the DNA binding region is the 100B mutation. 
     
     
         56 . The plasmid of  claim 50 , wherein the mutation in the DNA binding region is the 201C mutation. 
     
     
         57 . A prokaryotic recombinant host cell comprising a heterologous pir gene encoding a π protein and a DNA molecule comprising a heterologous gene encoding a protein, a selection gene, a target region for a site-specific recombinase, and a conditional origin of replication whose functionality in a prokaryotic host cell requires a π protein, wherein the heterologous pir gene comprises at least one mutation in the pir gene leucine zipper-like motif or the pir gene DNA binding region. 
     
     
         58 . The prokaryotic recombinant host cell of  claim 57 , wherein the heterologous pir gene comprises at least one mutation in the leucine zipper-like motif. 
     
     
         59 . The prokaryotic recombinant host cell of  claim 58 , further comprising a mutation in the pir gene DNA binding region. 
     
     
         60 . The prokaryotic recombinant host cell of  claim 57 , wherein the heterologous pir gene is in a plasmid. 
     
     
         61 . The prokaryotic recombinant host cell of  claim 57 , wherein the heterologous pir gene is in the genome of the host cell. 
     
     
         62 . The prokaryotic recombinant host cell of  claim 57 , wherein the mutation in the DNA binding region is the 114C mutation. 
     
     
         63 . The prokaryotic recombinant host cell of  claim 57 , wherein the mutation in the DNA binding region is the 100B mutation. 
     
     
         64 . The prokaryotic recombinant host cell of  claim 57 , wherein the mutation in the DNA binding region is the 201C mutation. 
     
     
         65 . A method for detecting a plasmid copy-up mutation comprising:
 (a) introducing at least one mutation into a target sequence;   (b) transforming the mutated target sequence into a host cell comprising a plasmid, wherein the plasmid comprises a nucleotide sequence encoding urolll methyltransferase and the copy number of the plasmid is effected by the target sequence;   (c) growing the host cell under conditions wherein the nucleotide sequence is expressed to produce a culture of host cells;   (d) exposing the culture of host cells to UV light; and   (e) detecting fluorescence produced by the culture of host cells.   
     
     
         66 . The method of  claim 65 , wherein the nucleotide sequence is the cobA gene from  Pseudomonas denitrificans.    
     
     
         67 . The method of  claim 65 , further comprising comparing the fluorescence detected in (e) with fluorescence produced by a culture of host cells comprising an non-mutated target sequence.