141. Development of a One-Step Column Purification of Minicircle Vector DNA Devoid of Bacterial Sequences That Results in High Level Persistent Transgene Expression In Vivo

Abstract

We have shown that episomal circular DNA vectors free of plasmid bacterial DNA sequences are capable of indefinite high level transgene expression in vivo. The minicircle vector is generated in bacteria from a parental plasmid containing an inducible phiC31 integrase gene, and att P and att B recognition sites flanking the therapeutic expression cassette. Recombination results in the formation of two circular DNA molecules, one containing the plasmid bacterial backbone and the other the eukaryotic expression cassette. Previously, the minicircle was purified away from the plasmid bacterial DNA by cesium chloride banding in an ultracentrifuge (Chen et al., Mol Ther. 8:495, 2003). We have now devised a one-step purification method that results in a high yield of minicircle vector. To do this, two copies of the phiC31 integrase gene under the control of an inducible promoter (BAD), together with the transgene expression cassette flanked by the attB and attP sites, were built into a minicircle-producing plasmid, p2phiC31. An intron encoded restriction enzyme I-Sce I gene, also under the control of the BAD promoter, together with an I-Sce I restriction site, were also built into p2phiC31. Top 10 E. coli was used to amplify p2phiC31 using standard plasmid preparation. To induce the phiC31 integrase-mediated minicircle formation, an over night culture was re-suspended 4 to 1 (vol/vol) in fresh LB broth containing the pBAD inducer 1% L-arabinose and incubated at 32°C with constant shaking for one hour. The temperature of the incubation was increased to 37°C, to optimize I-Sce I enzymatic activity, for an additional 2 hours. Linearization of the plasmid bacterial DNA circle by the restriction enzyme resulted in its rapid degradation by exonucleases in bacteria. The mini-circle expression cassette becomes the only episomal circular DNA and is easily isolated by an affinity purification column. Using this technology to prepare a minicircle encoding a 4 kb human factor IX expression cassette, > 1 mg of minicircle of 97 percent purity can be prepared from 1 liter of over night bacterial growth. When injected into the livers of animals by hydrodynamic infusion, about 10 μg/ml of serum hFIX was expressed for up to 7 weeks (length of study). The high yield, simple purification, and robust and persistence of transgene expression makes these vectors viable for in vivo gene therapy applications.