516. Development of a Minicircle Vector Free of Plasmid Bacterial DNA Sequences and Capable of øC31-Mediated Site-Specific Integration
Authors: Zhi-Ying Chen, Cheng-Yi He, Anja Ehrhardt, Hui Xu, Mark A. Kay
Journal: Molecular Therapy (2006)

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It has been well documented that plasmid bacterial DNA sequences (plasmid BB) can silence the expression of an episomal or integrated transgene. Current site-specific integrating technologies are hampered in part by the fact that the integrated transgene expression cassette is covalently linked with the plasmid BB. As a result, a substantial proportion of integrated transgenes are expressed at reduced levels or silenced. We have previously reported the development of a robust method for producing episomal minicircle vectors that are devoid of plasmid BB and capable of persistent and high level transgene expression in quiescent tissues in vivo. The minicircle was generated from a parental plasmid, which contained an expression cassette flanked with the short form recombination sites, sattB and sattP, two copies of the recombinase |[oslash]|C31 and one copy of the endonuclease I-Sce I genes under the control of the inducible promoter BAD. An I-Sce I site was engineered into the plasmid BB. Minicircle containing the therapeutic transgene was generated by the intramolecular recombination between the sattB and sattP sites mediated by the |[oslash]|C31 recombinase after the addition of the inducer L-Arabinose to the bacterial culture. A second DNA circle composed of plasmid BB sequences together with the I-Sce I site was also formed but was degraded after linearization by the I-Sce I endonuclease. This resulted in milligram yields of minicircle DNA from a liter of culture that was then purified by simple column chromatography. To extend the application of the minicircle to rapidly replicating cells, we elected to make a site-selective integrating form of this vector. To do this, we devised a strategy to include a functional sattB site into the minicircle. We inserted an additional sattB site between the sattP and the expression cassette, and replaced the original upstream sattB with a full length attB (LattB), resulting in the efficient recombination between the LattB and sattP, and a high yield of sattB-containing minicircle (sattB.MC). An in vitro study demonstrated that the sattB site is capable of mediating efficient integration. To test for in vivo integration, 10 |[mu]|g of the integrating sattB.MC expressing human factor IX (hFIX) was infused into mouse liver together with 20 |[mu]|g of a plasmid expressing either wild type or mutant |[oslash]|C31. Seven weeks later a surgical two-thirds partial hepatectomy (PH) was performed. The procedure induces liver regeneration resulting in the loss of about 95% of the episomal DNA. Nine weeks after PH, the mice receiving wild type and mutant |[oslash]|C31 expressed 40 and 15% of serum hFIX, respectively as compared to corresponding non-PH group. Southern blot analysis confirmed integration of attB.MC in the genome of mice receiving wild type |[oslash]|C31, but not mice treated with mutant |[oslash]|C31. These results suggest that the sattB.MC is capable of integration in vivo. Further studies are underway to establish the advantages of the integrating minicircle vector.