Gene Therapy (1999) 6 , 209–218  1999 Stockton Press All rights reserved 0969-7128/99 $12.00 http:/ / www.stockton-press.co.uk/ gt Minicircle: an improved DNA molecule for in vitro and in vivo gene transfer A-M Darquet 1 , R Rangara 1 , P Kreiss 1 , B Schwartz 1 , S Naimi 2 , P Delae `re 2 , J Crouzet 2 and D Scherman 1 1 UMR 133 CNRS/Rho ˆne-Poulenc Rorer and 2 Rho ˆne-Poulenc Rorer Gencell, Centre de Recherche de Vitry-Alfortville, Vitry sur Seine, France Minicircles are a new form of supercoiled DNA molecule for enhancer/promoter. Comparing maximal differences, these nonviral gene transfer which have neither bacterial origin of minicircles gave 2.5 to 5.5 times more reporter gene replication nor antibiotic resistance marker. They are thus activity than the unrecombined plasmid in the NIH3T3 cell smaller and potentially safer than the standard plasmids line and rabbit smooth muscle cells. Moreover, injection in currently used in gene therapy. They were obtained in E. vivo into mouse cranial tibial muscle, or human head and coli by att site-specific recombination mediated by the neck carcinoma grafted in nude mice resulted in 13 to 50 phage  integrase, which was used to excise the times more reporter gene expression with minicircles than expression cassette from the unwanted plasmid with the unrecombined plasmid or larger plasmids. Histo- sequences. We produced two minicircles containing the logical analysis in muscle showed there were more trans- luciferase or -galactosidase gene under the control of fected myofibers with minicircles than with unrecombined the strong human cytomegalovirus immediate–early plasmid. Keywords: gene transfer; gene therapy; DNA vaccine;  integrase Introduction Minicircles are new supercoiled DNA molecules for non- viral gene transfer, which have neither bacterial origin of replication nor antibiotic resistance gene. 1 Minicircles are obtained in E. coli by att site-specific recombination mediated by the phage  integrase. Minicircles may con- tain no more than a eukaryotic expression cassette and the attR fragment resulting from the attP/attB recombi- nation event. Thus they are a nondisseminating genetic material for nonviral gene therapy. Furthermore, mini- circles carry only short bacterial sequences. Such sequences may cause undesirable effects such as the pro- duction of antibodies against bacterial proteins expressed from cryptic upstream eukaryotic expression signals, 2 changes in eukaryotic gene expression caused by the anti- biotic resistance marker, 3 and immune responses to CpG sequences. 4–6 In this study, we have produced new minicircles carry- ing the luciferase or -galactosidase reporter gene under the control of the strong human cytomegalovirus immediate–early enhancer/promoter. The efficiency of gene transfer with these minicircles was compared with that of the corresponding unrecombined plasmid or larger plasmids, both in vitro in transformed primary cells, and in vivo, in muscle and experimental tumors. Our results suggest that the use of minicircles may be Correspondence: D Scherman, UMR 133 CNRS/Rho ˆne-Poulenc Rorer, Centre de Recherche de Vitry-Alfortville, 13, Quai Jules Guesdes, 94403 Vitry sur Seine, France Received 8 May 1998; accepted 16 September 1998 more efficient for in vitro and in vivo gene transfer than classical plasmids. Results Construction and production of CMV luc+ and CMV -gal minicircles The plasmids pXL3186 and pXL3187 contain the ColE1 origin of replication (ie they are derived from pBR322), a kanamycin-resistance gene, and, in the same orien- tation, the 385 bp attP site of  phage and the 31 bp E. coli minimal attB sequence 7 (Figure 1). In pXL3186, the cDNA encoding the modified firefly luciferase was inserted between the  attP and attB sites. In this 5.5 kb construct, the reporter gene is under the control of the CMV enhancer/promoter, fused to the herpes simplex virus thymidine kinase gene 5′ untranslated leader. The 5.5 kb construct also contains a polyadenylation signal from the bovine growth hormone gene. In pXL3187 the -galactosidase gene was cloned between the  attP and attB sites. In this 7.3 kb construct, the reporter gene is under the control of the CMV enhancer/promoter and has an SV40 polyadenylation signal. Recombination was achieved by thermal induction of  integrase at 42°C in E. coli D1210HP. 8 E. coli D1210, which lacks the thermosensitive lysogen, was used as a control. Phage  integrase mediates site-specific recombi- nation between the attP and attB sites. The two recombi- nation sites are in the same orientation on the same replicon. Recombination results in the excision of a super- coiled minicircle carrying only the eukaryotic gene expression cassette and the recombinant attR site. 9 Int-