DNA: REPLICATION REPAIR AND RECOMBINATION
An araC-controlled Bacterialcre Expression System to Produce DNA Minicircle Vectors for Nuclear and Mitochondrial Gene Therapy*

https://doi.org/10.1074/jbc.M010873200Get rights and content
Under a Creative Commons license
open access

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 vitroand in vivo applications whereas minicircle expressionin 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.

Cited by (0)

Published, JBC Papers in Press, April 13, 2001, DOI 10.1074/jbc.M010873200

*

This work was supported by the Medical Research Council, The March of Dimes Birth Defects Foundation, and the Association Française de Lutte Centre la Mucoviscidose.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.