Minicircle: an improved DNA molecule for in vitro and in vivo gene transfer

Minicircles are a new form of supercoiled DNA molecule for nonviral gene tr ansfer which have neither bacterial origin of replication nor antibiotic re sistance marker. They are thus smaller and potentially safer than the stand ard plasmids currently used in gene therapy. They were obtained in E. coli by att site-specific recombination mediated by the phage lambda integrase, which was used to excise the expression cassette from the unwanted plasmid sequences. We produced two minicircles containing the luciferase or beta-ga lactosidase gene under the control of the strong human cytomegalovirus imme diate-early enhancer/promoter. Comparing maximal differences, these minicir cles gave 2.5 to 5.5 times more reporter gene activity than the unrecombine d plasmid in the NIH3T3 cell line and rabbit smooth muscle cells. Moreover, injection in vivo into mouse cranial tibial muscle, or human head and neck carcinoma grafted in nude mice resulted in 13 to 50 times more reporter ge ne expression with minicircles than with the unrecombined plasmid or larger plasmids. Histological analysis in muscle showed there were more transfect ed myofibers with minicircles than with unrecombined plasmid.