Gene Therapy (1999) 6, 1482–1488 1999 Stockton Press All rights reserved 0969-7128/99 $12.00 http://www.stockton-press.co.uk/gt BRIEF COMMUNICATION pCOR: a new design of plasmid vectors for nonviral gene therapy F Soubrier, B Cameron, B Manse, S Somarriba, C Dubertret, G Jaslin, G Jung, C Le Caer, D Dang, JM Mouvault, D Scherman, JF Mayaux and J Crouzet Rhoˆne-Poulenc Rorer, Centre de Recherche de Vitry Alfortville, 13 Quai J Guesde, 94403 Vitry-sur-Seine, France A totally redesigned host/vector system with improved initiator protein, ␲ protein, encoded by the pir gene limiting properties in terms of safety has been developed. The its host range to bacterial strains that produce this trans- pCOR plasmids are narrow-host range plasmid vectors for acting protein; (2) the plasmid’s selectable marker is not an nonviral gene therapy. These plasmids contain a con- antibiotic resistance gene but a gene encoding a bacterial ditional origin of replication and must be propagated in a suppressor tRNA. Optimized E. coli hosts supporting specifically engineered E. coli host strain, greatly reducing pCOR replication and selection were constructed. High the potential for propagation in the environment or in yields of supercoiled pCOR monomers were obtained (100 treated patients. The pCOR backbone has several features mg/l) through fed-batch fermentation. pCOR vectors carry- that increase safety in terms of dissemination and selec- ing the luciferase reporter gene gave high levels of lucifer- tion: (1) the origin of replication requires a plasmid-specific ase activity when injected into murine skeletal muscle. Keywords: gene therapy; plasmid DNA; conditional replication; selection marker; multimer resolution Two different types of DNA vehicles, based criteria.4 These high copy number plasmids carry a mini- on recombinant viruses and bacterial DNA plasmids, are mal amount of bacterial sequences, a conditional origin used in gene therapy. Although virus-based vectors are of replication that prevents dissemination during widely used and very efficient vectors, the potential risks production or therapy and do not contain an antibiotic of viral gene delivery have to be seriously considered. E. resistance gene. coli plasmid-based vectors are non-infectious and versa- The pCOR backbone consists of three bacterial tile vehicles for gene therapy. The manufacture of thera- elements: the 0.4 kb R6K ␥ conditional origin of repli- peutic plasmid DNA that carries potent human genes cation (ori ␥), which requires the R6K ␲ initiator protein becomes a reality. Thus, new approaches that take into to be functional, a 0.2 kb selectable tRNA suppressor account safety and environmental consideration must gene (sup Phe), and a 0.4 kb cer (ColE1 resolution) be developed. fragment to resolve pCOR oligomers. Vector design is a major contributor to the safety of Such plasmids can only replicate in ␲-producing bac- production and therapy. Efforts to improve the plasmid teria considerably limiting their host range. pCOR selec- backbone have focused on the improvement of transgene tion uses expression of a synthetic amber suppressor expression1 or plasmid copy number2 but no rational tRNA gene, specific for phenylalanine (sup Phe), which design in terms of dissemination or size, has been does not require antibiotics. This suppressor corrects an reported. The ColE1-derived bacterial plasmids are cur- amber mutation in argE gene making it possible for the rently used in gene therapy experiments and clinical recombinant host strain to grow on a minimal medium trials. As far as the safety and containment of such gen- lacking arginine (Figure 1). etic material are concerned, two elements are deleterious: The pCOR conditional origin of replication comes from the ColE1 origin of replication and the antibiotic resist- a natural plasmid, R6K, rather than the widely used ance marker. Clinical use of such plasmids could poten- ColE1-derived plasmids. R6K is a 38 kb theta-replicating tially lead to their dissemination in the environment or R (resistance) plasmid. R6K replication functions5 are in the patient. This could be of the utmost importance for clustered in a 5.5 kb DNA fragment that contains three the immunodepressed or for cystic fibrosis patients who origins of replication: ␣ and ␤ that are mainly used, and suffer from chronic bronchopulmonary infections.3 pCOR ␥. All three are under the control of a single replication plasmids, for plasmid with conditional origin of repli- initiation protein, ␲, the product of the pir gene. This pro- cation, were specifically designed and developed for gene tein binds to direct nucleotide sequence repeats (seven therapy. These new plasmids are small efficient transgene tandemly associated 22-bp direct repeats) at the origin of carriers and fulfill several quality assurance and safety replication. It is a dual regulator, exerting both a positive effect on replication and negative feedback that maintains R6K at its characteristic copy number (15 per genome). Correspondence: J Crouzet Copy-up mutants inhibit replication less and maintain Received 21 December 1998; accepted 12 April 1999 the ␥ origin at a high copy number. pir1166 contains a pCOR plasmids for gene therapy F Soubrier et al 1483 Figure 1 pCOR host vector system. The XAC-1pir116 strain requires arginine for growth on minimal medium whereas the arginine deficiency of the pCOR host is corrected by the tRNA sup Phe from the plasmid. The pCOR plasmid, the pir116 and argEam genes, and the XAC-1pir116 chromosome ⌬ are not drawn to scale. The amber mutation is shown by a stop and its correction by a crossed-out stop. XAC-1pir116 genotype: ara (lac-proB)x111 ⌬ Ј + gyrA argEam rpoB thi uidA( MluI)::pir116 F (proB lacI373 lacZu118am). single base mutation (CCC→CTC) that leads to a proline Comparison of pXL2666 (ori ␥-KmR) and pXL2730 (ori to leucine substitution at position 106 in ␲. ␥-KmR-cer) topology (Figure 2) showed that cer leads to The minimum genetic information required to main- monomerization of R6K-derived plasmids. A deletion12 tain R6K derivatives is the 400-bp ␥ origin (ori ␥) and the leading to the inactivation of cer, was introduced into cis or trans-acting ␲ initiator protein.5 Ori ␥ was isolated pXL2666 to generate pXL2754 (ori ␥-KmR-⌬cer). This con- from the suicide vector pUT-T7pol7 as a 0.4 kb BamHI– struct led to the production of plasmid multimers. These EcoRI fragment and was ligated to the kanamycin resist- experiments were also carried out in XAC-1pir and XAC- ance gene (KmR) from pUC4K8 to generate pXL2666. Ori 1pir116 strains (data not shown) which differ only by the ␥ replication and plasmid copy number were assayed in pir allele. Thus, these results shed light on the link wild-type and copy-up backgrounds. pXL2666 was trans- between plasmid multimerization/copy-up pir mutations ferred into a pir (BW190949) and a pir116 strain and multimer resolution via cer-mediated intramolecu- (BW196109)ofE. coli. Agarose gel electrophoresis showed lar recombination. that there were five to 10 more plasmid DNA copies in An important original feature of pCOR is the absence the pir116 than in the pir strain, consistent with previous of an antibiotic resistance gene for selection in E. coli. E. work6 (data not shown). It also pointed out that the coli amber (TAG) suppressor tRNA genes do not contain monomer/oligomer ratio of R6K-derivatives was affected any sequences derived from phages or transposons and by the presence of the copy-up mutation which resulted are very small (less than 100 bp). They are efficient plas- in the major plasmid species being multimers, mostly mid selection markers, even in high-density cultures of dimers (Figure 2). Plasmid linearization by EcoRI indi- E. coli.13 A synthetic amber suppressor tRNA gene, spe- cated that these preparations contained a mixture of cific for phenylalanine (sup Phe) was used as the pCOR monomer and the corresponding oligomers. Topoisomer- marker. sup Phe is expressed from a strong constitutive ase treatment showed that the high molecular weight E. coli promoter, Plpp. This cassette (0.17 kb NarI–HindIII species were circular, supercoiled, plasmid multimers fragment) was isolated from pCT2-F14 and was tested in rather than catenanes. These plasmids are relaxed by E. coli XAC-1.14 The chromosomal argE gene of XAC-1 DNA topoisomerase I and topoisomerase IV10 and no contains an amber mutation that results in a truncated decatenation by topoisomerase IV was observed, and therefore inactive product. As argE encodes N-ace- although this enzyme was active on kinetoplast DNA tylornithinase which is essential for arginine biosynth- (data not shown). Plasmid multimerization is not com- esis, this strain cannot grow in the absence of arginine. patible with pharmaceutical use due to low lot to lot The suppressor tRNA sup Phe causes the correct trans- reproducibility and too few molecules per unit weight. lation of mRNA and the synthesis of an active full-length As well as reducing the number of independently segre- N-acetylornithinase. Thus, XAC-1 can grow on minimal gating molecules, it may also severely reduce plasmid medium lacking arginine as long as the plasmid is stability by affecting plasmid segregation during bacterial present, ensuring plasmid selection. However, this strain cell division. In our effort to resolve multimers, we tested does not synthesize the R6K ␲ initiator protein and thus the cer fragment that can promote monomerization inde- cannot replicate pCOR. Therefore, the ␲-encoding gene, pendently of the replicon involved. This cis-acting DNA pir (or the copy-up mutant pir116), was inserted into the determinant reduces multimerization via a site-specific XAC-1 chromosome by homologous recombination. This recombination event that requires only E. coli-encoded method does not introduce any mobile genetic element, proteins. Two recombinases, XerC and XerD, are neces- such as transposons or phages, that affect genome stab- sary, along with two accessory proteins, ArgR and PepA, ility.