Abstract
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.
Highlights
There is increasing evidence to suggest that plasmid DNA used for non-viral gene delivery can cause unacceptable inflammatory responses in eukaryotes [1,2,3,4,5]
By the simple expedient of removing the bacterial DNA complement, we significantly reduce the size and CpG content of these expression vectors, which should 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
Summary
There is increasing evidence to suggest that plasmid DNA used for non-viral gene delivery can cause unacceptable inflammatory responses in eukaryotes [1,2,3,4,5]. Removal of bacterial sequences needs to be efficient, using the smallest possible excision site, while creating supercoiled DNA minicircles, consisting solely of gene expression elements under appropriate mammalian control regions. This can be achieved by the use of Cre recombinase, a bacteriophage P1-derived integrase (9 –11), catalyzing site-specific recombination between direct repeats of 34 base pairs (loxP sites). In the case of a supercoiled plasmid containing DNA flanked by two loxP sites in the same orientation, Cre recombination produces two DNA molecules that are topologically unlinked, circular, and mainly supercoiled [10], each containing a single 34-bp loxP site
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