An araC-controlled bacterial cre expression system to produce DNA minicircle vectors for nuclear and mitochondrial gene therapy.
The presence of CpG motifs and their associated sequences in bacterial DNA causes an immunotoxic response following the delivery of these plasmid vectors into mammalian hosts. We describe a biotechnological approach to the elimination of this problem by the creation of a bacterial cre recombinase expression system, tightly controlled by the arabinose regulon. This permits the Cre-mediated and -directed excision of the entire bacterial vector sequences from plasmid constructs to create supercoiled gene expression minicircles for gene therapy. Minicircle yields using standard culture volumes are sufficient for most in vitro and in vivo applications whereas minicircle expression in vitro is significantly increased over standard plasmid transfection. By the simple expedient of removing the bacterial DNA complement, we significantly reduce the size and CpG content of these expression vectors, which should also reduce DNA-induced inflammatory responses in a dose-dependent manner. We further describe the generation of minicircle expression vectors for mammalian mitochondrial gene therapy, for which no other vector systems currently exist. The removal of bacterial vector sequences should permit appropriate transcription and correct transcriptional cleavage from the mitochondrial minicircle constructs in a mitochondrial environment and brings the realization of mitochondrial gene therapy a step closer.
Bigger
B W
BW
Cystic Fibrosis Gene Therapy Group, Division of Biomedical Sciences, SAF Bldg., Imperial College of Science, Technology and Medicine, Exhibition Rd., London SW7 2AZ, United Kingdom. b.bigger@ic.ac.uk
Tolmachov
O
O
Collombet
J M
JM
Fragkos
M
M
Palaszewski
I
I
Coutelle
C
C
D051777:AraC Transcription Factor; D001089:Arabinose / Q000235:genetics; D001426:Bacterial Proteins; D001483:Base Sequence; D002467:Cell Nucleus / Q000235:genetics*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics*; D015316:Genetic Therapy; D005822:Genetic Vectors; D019426:Integrases / Q000235:genetics*; D008928:Mitochondria / Q000235:genetics*; D012097:Repressor Proteins / Q000235:genetics*; D014157:Transcription Factors; D014764:Viral Proteins
D051777:AraC Transcription Factor; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D012097:Repressor Proteins; D014157:Transcription Factors; D014764:Viral Proteins; D001089:Arabinose; C045073:Cre recombinase; D019426:Integrases