A robust system for production of minicircle DNA vectors

A minicircle episomal DNA vector is a circular expression cassette devoid of the bacterial plasmid DNA backbone. These vectors have been used for years in preclinical gene transfer research because of their 10to 1,000-fold enhancement compared with regular plasmids in long-term transgene expression in quiescent tissues in vivo1,2 and in vitro3. The mechanism of enhanced transgene expression is unclear but may result from eliminating heterochromatin formation induced by the plasmid backbone4 and/or from reducing the death of transfected cells from inflammation due to CpG responses when plasmids are delivered by lipid carriers5. The major obstacle to widespread use of minicircles has been their time-consuming, labor-intensive production. In our previous minicircle production schemes (Fig. 1a), the minicircle producer plasmid contained a transgene expression cassette flanked with attB and attP, a set of inducible enzyme genes (a gene encoding homing endonuclease I-SceI and two copies of the gene encoding ΦC31 integrase) and an I-SceI recognition site6. The attB and attP sites are the bacterial and phage attachment sites of ΦC31 integrase, and the ΦC31 and I-SceI genes are regulated by the l-arabinose–inducible araCBAD system. Minicircle DNA is generated by recombination between the attB and attP sites, and I-SceI initiates the destruction of the plasmid DNA backbone circle by cutting through the engineered I-SceI site (Fig. 1a). Although the yields from this protocol were ~1 mg of minicircle DNA from 1 liter of overnight culture, the preparations still contained ~3–15% of the input minicircle producer plasmid plus the plasmid backbone circle as contaminants. Including CsCl equilibrium gradient centrifugation to remove these unwanted DNAs, the production procedure is four labor-intensive days longer than routine plasmid production protocols. Other groups have made minicircle DNA vectors using different recombinases, such as the bacteriophage λ integrase7 or Cre recombinase8. Limitations of these a robust system for production of minicircle Dna vectors