(1997) 4, 1341–1349  1997 Stockton Press All rights reserved 0969-7128/97 $12.00 A new DNA vehicle for nonviral : supercoiled minicircle

A-M Darquet1, B Cameron2, P Wils1, D Scherman1 and J Crouzet2 1UMR 133 CNRS/Rhoˆne-Poulenc Rorer and 2Rhoˆne-Poulenc Rorer Gencell, Centre de Recherche de Vitry-Alfortville, 13, Quai Jules Guesdes, 94403 Vitry sur Seine, France

Plasmids currently used for nonviral gene transfer have the excised in vivo after thermoinduction of the integrase gene disadvantage of carrying a bacterial origin of replication leading to the formation of two supercoiled molecules: the and an antibiotic resistance gene. There is, therefore, a minicircle and the starting lacking the expression risk of uncontrolled dissemination of the therapeutic gene cassette. In various cell lines, purified minicircles exhibited and the antibiotic resistance gene. Minicircles are new a two- to 10-fold higher luciferase reporter gene activity DNA delivery vehicles which do not have such elements than the unrecombined plasmid. This could be due to either and are consequently safer as they exhibit a high level of the removal of unnecessary plasmid sequences, which biological containment. They are obtained in E. coli by att could affect , or the smaller size of mini- site-specific recombination mediated by the phage ␭ inte- circle which may confer better extracellular and intracellular grase. The desired eukaryotic expression cassette bioavailability and result in improved gene delivery bounded by the ␭ attP and attB sites was cloned on a properties. recombinant plasmid. The expression cassette was

Keywords: DNA ; gene therapy; site-specific recombination; ␭ integrase; gene expression; DNA

Introduction clearly undesirable for other applications for which an immune stimulation is not required. currently used in preclinical and clinical trials The aim of this work was to develop safer genetic of nonviral gene transfer contain a prokaryote origin of material for nonviral gene therapy. We produced super- replication and an antibiotic resistance marker, both of coiled recombinant DNA molecules, called minicircles, which are undesirable. Clinical use would lead to the dis- which contain only the therapeutic expression cassette. semination of prokaryotic replicative recombinant DNA These minicircles are the product of in vivo excision of in patients. Endogenous Enterobacteriacae, in which such the desired cassette by site-specific recombination plasmids replicate, may acquire the recombinant DNA between the two attP and attB sequences driven by E. coli resulting in an uncontrolled dissemination of the thera- bacteriophage ␭ integrase. We obtained and purified a peutic and antibiotic resistance genes. These recombinant minicircle carrying a luciferase expression cassette (luc- bacteria might then have a selective advantage, parti- minicircle). These minicircles have no plasmid backbone cularly if the corresponding antibiotic is administered. sequences and thus avoid the deleterious effects of proka- Moreover, prokaryotic sequences can have other unde- ryotic sequences present in the plasmid. In addition, sirable effects. For instance have been pro- these recombinant molecules presented gene transfer ␦ duced against prokaryotic -endotoxin after injection of properties superior to those of standard plasmids. There- ␦ a plasmid carrying the -endotoxin fore, minicircles are a promising alternative to plasmid gene, expressed from cryptic upstream eukaryotic DNA for nonviral gene therapy in terms of biosafety, 1 expression signals. Expression of the neomycin resist- improved gene transfer and potential bioavailability, due ance marker alters the expression of genes in mammalian to their minimal size. cultured cells.2 Furthermore, elements in the plasmid backbone such as the ampicillin-resistance gene (AmpR) result in a lower efficiency of expression.3 In Results addition, short immunostimulatory DNA sequences, con- taining CpG dinucleotides, present in the prokaryotic Construction of pXL2650 backbone of plasmids, function as adjuvants for the Plasmid pXL2650 carries the Photinus pyralis luciferase immunogenic properties of plasmids encoding antigens.4 gene (luc) expression cassette (luc-cassette). This con- This DNA adjuvant effect would be valuable for gene struct is 7.4 kb and has the following components (Figure immunization, or gene immunotherapy for cancer, but is 1): the ColE1 origin of replication (ie a pBR322-derived plasmid), the E. coli galK gene, an ampicillin resistance gene, the 530 bp attP sites of ␭ phage and the 31 bp E. coli Correspondence: J Crouzet minimal attB sequence5 in the same orientation, and the Received 11 April 1997; accepted 31 July 1997 luc-cassette from pGL2 control (Promega, Madison, WI, Supercoiled DNA molecules for nonviral gene transfer A-M Darquet et al 1342 USA). Between the ␭ attP and attB sites the luc reporter gene is under the control of the SV40 early , and upstream from the intron of the SV40 small-t antigen, the SV40 early polyadenylation signal and an enhancer from the SV40 early promoter.

Production of luc-minicircle The attP and attB sites are two short fragments of the phage ␭ and the E. coli chromosome, respect- ively. Phage ␭ integrase mediates the integration of the circular phage genome in the E. coli chromosome by recombination. This reaction needs two host proteins (FIS and IHF)5–7 in addition to the integrase. If the two att sites are in the same orientation on the same replicon, the recombination will result in the excision of a supercoiled molecule.8 Thus, Int-mediated recombination of pXL2650 (Figure 1) will lead to a minicircle with a luc-cassette of 3.4 kb and a 4 kb pXL2650-derivative containing the ori- gin of replication, the antibiotic marker gene and the recombinant site attL termed a miniplasmid. The mini- circle carries only the reporter gene, with eukaryotic expression signals, and the recombinant site attR (Figure 1). The E. coli strain D1210HP was used in this study; it is a derivative of D1210 and harbors a ␭ thermosensitive lysogen (xis kil− cI857) defective for lethal and lytic func- tions of the prophage thus preventing cell lysis.9 Upon thermal shift to 42°C the induction of a lytic cycle occurs and the integrase is produced resulting in the Int- mediated reaction. However, the reverse reaction requir- ing the Xis protein does not occur since the lysogen is xis−. Subsequently, the E. coli DNA topoisomerase IV will decatenate the two products of the reaction.10 Plasmid DNA extracts from D1210HP pXL2650 cul- tures shifted from 30°Cto42°C contained the expected resolution products of pXL2650 (Figure 2a), ie the luc- minicircle and the miniplasmid. Restriction digests with HindIII, NsiI and SphI which cut only the luc-minicircle at one, two and three sites, respectively, gave the expected fragments and left the miniplasmid uncut (Figure 2b). AlwNI, MluI, SnaBI, ScaI and XmnI for which there are restriction sites only in the miniplasmid gave the expected patterns of digestion leaving the luc-minicircle undigested (Figure 2b). This recombination was not observed in D1210HP pXL2650 cultured at 30°Corin D1210 harboring pXL2650 indicating it was Int-mediated (Figure 2a). Thus Int can mediate in vivo excision of a therapeutic gene expression cassette. The yield of unre- combined plasmid was around 40% of the starting material indicating that the reaction has to be improved.

Purification of the luc-minicircle Figure 1 pXL2650 restriction map and scheme of in vivo site-specific Supercoiled molecules obtained after recombination in recombination of pXL2650. The restriction map of pXL2650 is shown at the top of the figure. (a) Pairing of the two att sites of phage ␭ (arrows D1210HP pXL2650 were digested with XmnI and AlwNI, indicate their relative orientations). (b) The att sites recombine in the bac- both of which cut only the miniplasmid and pXL2650 teriophage ␭ integrative reaction, which is catalyzed by the ␭ integrase (Figure 1). Supercoiled molecules were then isolated by and requires the E. coli proteins IHF and FIS. (c) Recombination resolves CsCl-ethidium bromide (CsCl-EtBr) density gradient cen- pXL2650 into two molecules: one is a plasmid containing the origin of trifugation. Analysis by agarose gel electrophoresis replication and the ampicillin resistance gene (miniplasmid), the other is showed that the supercoiled luc-minicircle preparation the luc-minicircle. E. coli DNA topoisomerase IV is required for the decat- was free of pXL2650 and the miniplasmid (Figure 3). enation of the two resolved molecules. A, AlwNI; H, HindIII; M, MluI; Ns, NSiI; S, SnaBI; Sc, ScaI; Sp, SphI; Xb, XbaI; X, XmnI; ori, ColE1- Restriction digest analysis confirmed that the preparation derived origin of replication; luc, luc-cassette; galK, E. coli galK gene; contained the luc-minicircle (data not shown). AmpR, ampicillin resistance gene. Horizontal striped arrow, AmpR; verti- However, two minor bands (A and B) with lower cal striped arrow, luc; black square arrow, attP and attB sequences; waved electrophoretic mobilities were also observed in the luc- arrow, galK gene; white box, SV40 small-t antigen intron; black box, minicircle preparation (Figure 3). HindIII digestion of the SV40 early polyadenylation signal; hatched box, SV40 enhancer from early promoter; dotted box, SV0 early promoter. supercoiled preparation gave a single DNA band of Supercoiled DNA molecules for nonviral gene transfer A-M Darquet et al 1343

Figure 2 Production of luc-minicircle in D1210HP. (a) Analysis of the Int-mediated reaction in D1210 HP pXL2650. Plasmid pXL2650 was introduced into two E. coli strains, D1210 and D1210HP. D1210HP is D1210 carrying the ␭ xis− kil − cI857 phage lysogen. Upon thermal induction at 42°C, ␭ integrase is produced in D1210HP and the luciferase cassette is excised giving a luc-minicircle. Lanes 1 and 6: supercoiled DNA ladder; lane 2: plasmid DNA extracts from D1210HP pXL2650 after a thermal shift to 42°C; lane 3: uninduced D1210HP pXL2650; lane 4: D1210 pXL2650 shifted to 42°C and lane 5: uninduced D1210 pXL2650. Sizes in kb of the supercoiled DNA ladder are indicated on the left. Arrows A and B correspond to supercoiled minicircle and supercoiled miniplasmid, respectively. (b) Restriction digest of extrachromosomal molecules from induced D1210HP pXL2650. Undigested extrachromosomal molecules from D1210HP pXL26520 after induction (lane 1) and digested with AlwNI (lane 2), XmnI (lane 3), ScaI (lane 4), SnaBI (lane 5), MluI (lane 6), HindIII (lane 9), SphI (lane 10) and NsiI (lane 11). Indicated by an arrow: A, supercoiled minicircle; B, supercoiled miniplasmid; C, supercoiled pXL2650; D, linearized minicircle; E, linearized miniplasmid; F, linearized pXL2650. Supercoiled DNA ladder (lane 8) and linear DNA ladder (lane 7).

3.4 kb (Figure 3) the minor DNA being totally parison with supercoiled DNA molecular weight stan- digested. Similar results were obtained with other restric- dards (Figure 3). A 475 bp, 32P-labeled, AmpR gene probe tion cutting the luc-minicircle, whereas ScaI hybridized with both pXL2650 and miniplasmid, but not does not linearize this DNA preparation. The extra bands with DNA bands A and B. However, both bands A and may thus correspond with dimers and nicked luc-mini- B hybridized with a 3 kb 32P-labeled probe for the luc- circles (supercoiled plasmid DNA preparations are often cassette. Luc-minicircle preparations were also analyzed contaminated by both nicked and dimer forms of the after treatment with E. coli DNA topoisomerases I, II and plasmid). Indeed, the mobility of band A corresponded IV. Topoisomerase II (gyrase) supercoils relaxed mol- with that of the luc-minicircle dimer as estimated by com- ecules, whereas topoisomerase I and IV relax supercoiled Supercoiled DNA molecules for nonviral gene transfer A-M Darquet et al 1344

Figure 3 Analysis of the purified luc-minicircle. Lanes 1 and 8: supercoiled DNA ladder; lanes 2 and 7: linear DN ladder; lane 3: HindIII-digested and lane 4 undigested purified luc-minicircle; lane 5: plasmid DNA preparation from induced, and lane 6 uninduced, D1210HP pXL2650. In lane 4 bands A and B correspond to dimer and nicked luc-minicircle, respectively. Sizes in kb of the supercoiled and linear DNA ladders are indicated on the left and on the right, respectively.

molecules. DNA from band A and luc-minicircle DNA depended not only on the molarity of the luc-cassette but were relaxed by topoisomerase I and IV, but gyrase had also on the amount of DNA used. Similar results have no effect (data not shown). This indicates that the DNA been reported by Barthel et al.12 We found that there was in band A corresponds with supercoiled luc-minicircle a linear relationship between the amount of pXL2650 dimer and that the luc-minicircle is supercoiled. The elec- used to transfect NIH 3T3 cells and the resulting lucifer- trophoretic mobility of DNA in band B was not affected ase activity (Figure 4) but only when the total amount of by the action of any topoisomerase indicating that it was DNA added was fixed (1 ␮g), plasmid pBluescript II KS nicked DNA, presumably nicked luc-minicicle generated being used as carrier DNA. This plasmid contains only during the purification and storage of the supercoiled prokaryotic DNA and presumably does not interfere with preparation. Therefore, the purified luc-minicircle the eukaryotic machinery and luc-cassette preparation was indeed free of miniplasmid and expression. unrecombined plasmid. When no carrier DNA was added, the relationship between the pXL2650 concentration and luciferase Influence of total DNA concentration on transfection activity was not linear: there was a lag in the response, efficiency ie low transfection efficiency at low DNA concentration We developed a transfection protocol allowing the com- (0.1 and 0.2 ␮g per assay). Therefore, to compare the parison of gene expression from of different mol- transfection efficiencies of supercoiled molecules of dif- ecular weights. ferent sizes, carrier DNA was used so that the total Transfection experiments were performed using the amount of DNA was the same in each assay. DNA transfer agent RPR 120535 which consists of a dioct- adecyl lipid moiety linked to a spermine head group via Luciferase activities from luc-minicircle transfected cells a primary amine to generate a linear structure. This lipo- The potency of luc-minicircle for luciferase gene polyamine has transfection properties in the same expression was studied by with 1 ␮gof range as those of standard cytofectins.11 Luc gene pXL2650, 0.81 ␮g of pGL2 control or 0.46 ␮g of luc-mini- expression efficiencies for luc-minicircle (3.4 kb), pGL2 circle per assay with a complement of pBluescript KS II control (6 kb) and pXL2650 (7.4 kb) should ideally be to 1 ␮g as necessary. The molarity of the luc-cassette was compared using the same quantity of luc-cassette, and thus the same for all tests. Three charge ratios (3, 6 and thus differing amounts of total DNA. However, in pre- 9) of RPR120535 to DNA were assayed. liminary experiments the efficiency of transfection Five different cell lines were studied: NIH 3T3 (murine Supercoiled DNA molecules for nonviral gene transfer A-M Darquet et al 1345 Influence of plasmid size on transfection efficiency To confirm that the size of the supercoiled molecule affects the efficiency of gene transfer, we constructed a large (15.3 kb) plasmid, named pXL3067, containing the luc-cassette. This plasmid has a backbone derived from pRK41515 and the same luc-cassette as pGL2 control and pXL2650. Cells were transfected with 1 ␮g of pXL3067, 0.49 ␮g of pXL2650, 0.39 ␮g of pGL2 control or 0.22 ␮g of luc-minicircle (equivalent molarity of luc-cassette) made up to 1 ␮g with pBluescript KS II. Transgene expression in H460, NIH 3T3 and RSM cells transfected with pXL3067 was 19-, 76- and 545-fold lower, respect- ively, than in the same cell lines transfected with luc- minicircle (Figure 6). These results indicate that the larger the transfected supercoiled molecule, the lower the trans- fection efficiency.

Discussion Figure 4 Influence of total DNA concentration on gene expression. Com- parison of transfection efficiencies with different amounts of pXL2650, made We constructed and studied new DNA supercoiled mol- up (࡯)ornot(̆) to a total of 1 ␮g of DNA with pBluescript KS II and ecules called minicircles which can be used for nonviral complexed with RPR 120535 at a charge ratio of 3. Results are expressed in gene transfer. They have no bacterial origin of replication RLU/␮g of protein. T bars indicate the standard deviation (s.d.). or antibiotic resistance marker and are thus safer than plasmids currently used in preclinical and clinical trials. The probability that minicircles would be disseminated following their use for gene therapy is low due to the fibroblast), H460 (human nonsmall cell lung carcinoma), lack of bacterial replication origin. We cannot exclude the 3LL (mouse Lewis lung carcinoma), human aortic smooth possibility that an E. coli lysogenized by ␭ phage could muscle (HSM), and rabbit aortic smooth muscle (RSM) integrate the minicircle into its genome, through homolo- cells. Human smooth muscle cells are interesting for gene gous recombination between attR sites. However, this transfer purposes because of their proximity to the lumen would require two low probability events: the presence surface and their abundance in the vessel wall.13 The of an E. coli ␭ lysogen and . strength of reporter gene expression (highest to lowest) The same is true for standard plasmid antibiotic resist- was RSMC, NIH 3T3, 3LL, H460 to HSMC. This is not ance genes, whose homologous genes could be carried surprising as expression of reporter genes in HSMC is not only by E. coli, but also many other bacteria. We show much lower than that obtained in cell lines commonly that minicircles have higher transfection efficiencies than used for transfection, such as NIH 3T3 fibroblasts, commonly used plasmids and much higher efficiencies especially with the SV40 early promoter.14 The results of than larger plasmids. The differences were partly cell the transfection experiments are shown in Figure 5, with dependent, the largest differences being observed with luciferase activities normalized for total protein content. RSM and HSM cells. No activities above background were detected in lysates An alternative strategy to obtain minicircles is to use of cells transfected with DNA in the absence of cationic involving either a recombinase, or lipid (data not shown). Luc-minicircles expressed lucifer- restriction digestion followed by ligation. However, such ase activity in all the cell types tested. The activities, mea- approaches would be difficult to scale up for industrial sured as relative light units (RLU), were two- to four-fold use whereas in vivo recombination is simple and only (3LL and NIH 3T3), five- to seven-fold (H460) and 10-fold needs the production of recombinase to be induced. (RSM) higher in tested cells transfected with the luc- The backbone sequences in the luc-minicircle are only minicircle, than with the unrecombined plasmid 290 bp long, shorter than the 2.5–3 kb16 and 2 kb17 of the pXL2650. In HSM cells no significant luciferase activity backbones in standard and improved plasmids used in was detected following transfection with pXL2650, the clinic for nonviral gene transfer. Overall, minicircles whereas transfection with luc-minicircle gave 840 are about half the size of standard plasmids assuming the RLU/␮g of protein, the background being lower than transgene is shorter than 1 kb (the case for growth factors 6 RLU/␮g of protein. Transfection with the pGL2 control and , for example) and expressed under the con- plasmid led to luciferase activities similar to those trol of short viral expression signals such as the CMV obtained with pXL2650, except in H460 and RSM cells immediate–early promoter and the SV40 polyadeny- where the values were intermediate between those of luc- lation signals. minicircle and pXL2650. One limitation of the efficiency of in vivo nonviral gene These findings were confirmed with three independent delivery has been attributed to low extracellular and preparations of luc-minicircle, pGL2 control and pXL2650 intracellular bioavailability of the delivered complexes or DNA, showing that they were not a preparation effect. plasmids. As minicircles are smaller than standard plas- The experiments were reproduced between two and four mids, under particular conditions of complex formation, times in each cell line, giving the same ratio of RLU complexes between cationic lipids and minicircle could between supercoiled molecules. Moreover, in NIH 3T3 a be smaller than those obtained with standard plasmids, dose–response study showed that the observed effect was assuming that complexes form between one DNA mol- conserved at least from 1 to 0.5 ␮g of DNA per well. ecule and the transfecting agent. Small complexes should Supercoiled DNA molecules for nonviral gene transfer A-M Darquet et al 1346

Figure 5 Comparison of luciferase activities following transfection of various cell types with luc-minicircle, pGL2 control, or pXL2650 complexed with RPR120535. Transfections were performed at various charge ratios (3, 6 and 9) with the same molarities of luc-cassette and the same amount of total DNA (1 ␮g). Results are given in RLU/␮g of protein for NIH 3T3 fibroblasts (a), 3LL carcinoma cells (b), H460 carcinoma cells (c), RSM cells (d) and HSM cells (e). T bars indicate the standard deviation (s.d.). Supercoiled DNA molecules for nonviral gene transfer A-M Darquet et al 1347 bioavailability, again dependent on the size of the deliv- ered material. Indeed, DNA migration from the cyto- plasm to the nucleus appears to be a crucial step for suc- cessful gene transfer.19 It has been shown that the rate of protein transport from the cytoplasm to the nucleus is influenced by the number of nuclear localization sequences per protein, and most importantly by the size of the protein.22 Similarly, the size of transfected DNA molecules may determine their diffusion and/or trans- port to the nuclear pore. It has been reported that the percentage of ␤-galactosidase-positive myotubes follow- ing injection of plasmid DNA carrying the lacZ gene increases as the site of injection is closer to the nucleus,23 indicating that the diffusion of the plasmid from a site in the cytoplasm distant to the nucleus may be limiting.23 The efficiency of gene expression from linear and supercoiled DNA is different. Buttrick et al24 showed that luciferase activity is 50- to 100-fold greater for super- coiled plasmids than for linear forms. In our experiments the topology of transfected DNA molecules was investi- gated by agarose gel electrophoresis. Luc-minicircle, pGL2 control, pXL2650 and pXL3067 preparations con- tained similar ratios of relaxed to supercoiled forms. Dif- ferences in the topology of the transfected DNA mol- ecules are thus unlikely to be the reason for the differences of reporter gene activities observed. The enhanced gene delivery properties of minicircles may be due to absence of plasmid sequences, since plas- mid backbone elements have been shown to have an effect on gene expression. The replacement of an ampicil- lin resistance gene by a kanamycin gene led to a two- fold increase in luciferase activity.3 In our experiments, although all the molecules tested carry the same luc-cas- sette, it is surrounded by different sequences that might interfere with expression. For instance, small differences in supercoiling of the molecules, the presence of cryptic sites for the binding of transcription factors or short bac- terial immunostimulatory sequences which have been shown to interfere with gene expression by stimulating IFN-␣4 production might be responsible for all or part of the observed effects. The luc-minicircle was purified on density gradients, a method which is not acceptable for purification of clinical grade material. Only chromatographic methods are suit- able for preparing clinical grade DNA but they would not allow the separation of minicircle from miniplas- mid.25 We have developed an affinity chromatographic Figure 6 Influence of the size of supercoiled DNA on gene expression technique for the purification of plasmid DNA based on from the luc-cassette. Results are presented in RLU/␮g of protein for four the sequence-specific formation of a triple helix between different supercoiled molecules (luc-minicircle, pGL2 control, pXL2650 an immobilized and a specific sequence and pXL3067) at three charge ratios of RPR120535 to DNA with three present on the plasmid.26 This method could easily be cells lines: NIH 3T3 fibroblasts (a), H460 carcinoma cells (b) and RSM applied to separate minicircles from miniplasmids by cells (c). T bars indicate the standard deviation (s.d.). a short homopurine sequence next to the trans- gene cassette, thus allowing the specific triple helix inter- have better characteristics than larger complexes mainly action between minicircle and an appropriate because their diffusion coefficient is higher: the diffusion oligonucleotide covalently coupled to a chromato- coefficient is inversely proportional to the molecular graphic support. weight of the complex.18 The yield of recombination would have to be close to Complex size also plays a role in endocytosis, the 100% to avoid contamination with unrecombined plas- mechanism by which complexes enter the cell.19,20 This mid during affinity chromatography. This could be step is limited by the size of particles that can be taken achieved by optimizing the culture conditions of the up21 and complexes of diameter greater than 200 nm are recombinant E. coli and by overexpressing int. If these not efficiently taken up by the endocytic pathway. approaches are unsuccessful, affinity chromatography Another possible advantage of minicircles is better specific for sequences present in the miniplasmid and the Supercoiled DNA molecules for nonviral gene transfer A-M Darquet et al 1348 unrecombined plasmid would allow elimination from the were grown overnight at 30°C in LB Ap, diluted 1/100 in ° minicircle preparation. LB Ap and incubated at 30 CuntiltheOD610 reached 2. The ␭ phage lytic cycle was induced by a thermal shift to 42°Cfor10min.Thecultureswerefurtherincubatedfor Materials and methods 30 min at 30°C and bacteria then harvested. Extra chromosomal DNA was purified from these cells Standard DNA manipulation by a standard plasmid DNA purification technique,27 All standard manipulations, including digestion by using CsCl-EtBr density gradients. The purified DNA restriction enzymes, purification of DNA fragments by was then digested with AlwNI and XmnI. A second den- electroelution, electrophoresis of DNA, E. coli transform- sity gradient allowed the separation of the luc-minicircle ation, purification of supercoiled DNA by a CsCl-EtBr from the linearized miniplasmid and pXL2650. density gradient, and Southern blot analysis were perfor- Luc-minicircle preparations were concentrated 10-fold 26 med according to previously described techniques. with an ultrafree-15, Biomax 30 centrifugal filter device Intermediate plasmid constructions were introduced into (Millipore, Saint Quentin, France) according to the manu- ␣ ⌽ ⌬ ⌬ E. coli DH5 [F-, 80lacZ M15, (lacZYA-argF)U169, facturer’s recommendations. Luc-minicircle preparations ␭ deoR, TecA1endA1, hsdR17, phoA, supE44, - thi-1, gyrA96, were analyzed by the action of purified E. coli topoisom- relA1] (Clontech Laboratories, Palo Alto, CA, USA). DNA erases I, II and IV according to described techniques.10,32 concentrations were measured by reverse phase HPLC The 3 kb BamHI–BglII fragment from pGL2 control con- × analysis, using a Poros R2/H column (100 4.6 mm; Per- taining the luc gene and the 475 bp XmnI–BglII fragment Septive Biosystems, Cambridge, MA, USA) according to from the AmpR gene of pNH16a were 32P-labeled using described methods.26 the rediprime kit (Amersham). Restriction enzymes and T4 DNA ligase were pur- chased from Biolabs (New-England Biolabs, Beverly, MA, In vitro transfection protocol USA), Gibco-BRL (Life Technologies SARL, Cergy Pon- Cells were maintained in appropriate medium at 37°C toise, France) or Amersham (Amersham, Les Ulis, under a 5% CO2 humidified atmosphere. Dulbecco’s France). were synthesized on an modified Eagle’s medium (DMEM) (Gibco-BRL) sup- 28 Applied Biosystems 394 DNA/RNA synthesizer plemented with 100 units/ml of penicillin, 100 units/ml (Perkin Elmer, Courtaboeuf, France) and purified as pre- of streptomycin, 20 mml-glutamine and 10% fetal calf 29 viously described. Supercoiled and 1 kb linear DNA serum (Gibco-BRL) was used for NIH 3T3 (ATCC, Rock- ladders were from Gibco-BRL and Promega, respectively. ville, MD, USA), 3LL,25 H460 cells.33 RSM cells were obtained as previously described34 and were grown in Plasmid construction the same medium as above except that 20% fetal calf Plasmid pXL2648 was constructed from pNH16a9 which serum was used. contains the attP site of bacteriophage ␭. The attB site was Smooth muscle cell basal medium (Clonetics, San introduced into pNH16a using oligonucleotides, 5476 Diego, CA, USA) supplemented with 5 mg/l bovine insu- (5Ј-AATTGTGAAGCCTGCTTTTTTATACTAACTTGA- lin, 50 mg/l gentamicin sulfate, 50 ␮g/l amphotericin-B, GCGG-3Ј) and 5477 (5Ј-AATTCCGCTCAAGTTAGTA- 0.5 ␮g/l human , 2 ␮g/l human TAAAAAAGCAGGCTTCAC-3Ј), which reconstitute the fibroblast growth factor and 10% fetal calf serum minimal attB sequence.5 The two oligonucleotides were (Clonetics) was used for HSM cells (AOSMC 2708; hybridized and ligated into the EcoRI site in pNH16a Clonetics). Confluent cells were trypsinized and seeded giving plasmid pXL2648 which has an EcoRI site between in 24-well microtiter plates. Cells were transfected at 60– the attP and attB sequences which are in the same 80% confluence, which corresponds to 18 h after seeding orientation. for NIH 3T3, 3LL, H460, RSM cells and 1 week after seed- Plasmid pXL2649 was constructed by inserting the ing for HSM cells. The medium was changed every day KmR gene cassette of pUC4KIXX (Pharmacia, Uppsala, during the week before the transfection of HSM cells. Sweden) into the EcoRI site in pXL2648. The presence of Equal volumes of stock solution (10 mm solution in BamHI restriction sites on either side of the KmR cassette water obtained after heating for 30 min at 50°C) of 11 allowed the of the 3.15 kb BamHI–luc–BglII frag- RPR120535 (formula: H2N (CH2)3NH(CH2)4NH(CH)2)3 ment of pGL2-control (Promega), giving plasmid NHCH2 CONHCH2CON[(CH2)17-CH3]2] and supercoiled pXL2650, containing the luc-cassette between the att sites. DNA solution in 150 mm NaCl were mixed and incu- Plasmid pXL3040 corresponds to pGL2-control in bated for 10 min at 20°C. Cells were transfected with which the 1.6 kb BamHI–KmR-BamHI fragment of either pXL2650, pGL2 control, luc-minicircle or pXL3067. pUC4KIXX was inserted into the BglII site. Plasmid The total amount of DNA added to each well was pXL3067 was constructed in inserting the 5.1 kb BamHI– adjusted to 1 ␮g with pBluescript KS II (Stratagene Clon- luc–KmR–NheI fragment from pXL3040 between the XbaI ing System, La Jolla, CA, USA). Cells in 24-well microtiter and BamHI sites in pRK415,15 leading to a 15.3 kb plasmid plates were washed before transfection with 500 ␮lof harboring the same expression cassette as pXL2650. serum free-medium, once for HSM and RSM cells and twice for NIH 3T3, 3LL and H460 cells. Transfections Production of luc-minicircle were performed in 500 ␮l of serum-free medium by E. coli was grown in LB medium supplemented with adding 50 ␮l of lipid per DNA solution to each well. Each 50 ␮g/ml of ampicillin (LB Ap). Plasmid pXL2650 was transfection experiment was performed in triplicate. The introduced into two E. coli strains, D1210 [F-hsdS20, supE44, media were supplemented 2 h after transfection with ara-14, galK2, proA2, leuB6, rpsL20, xyl5, mtl1, recA, mcrB, either 10% of the appropriate serum for NIH 3T3, 3LL, ⌬(mcrC-mrr), lacIQ]30 and D1210HP (D1210 lysogenized H460 and HSM cells, and 20% for RSM cells. Luciferase with the ␭ cI857 xis− kil− phage31). The transformed strains activity was assayed 48 h later. Supercoiled DNA molecules for nonviral gene transfer A-M Darquet et al 1349 Luciferase activity 11 Byk G, Dubertret C, Schwartz B, Scherman D. 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