pFARs, plasmids free of antibiotic resistance markers, display high-level transgene expression in muscle, skin and tumour cells.
BACKGROUND
Nonviral gene therapy requires a high yield and a low cost production of eukaryotic expression vectors that meet defined criteria such as biosafety and quality of pharmaceutical grade. To fulfil these objectives, we designed a novel antibiotic-free selection system.


METHODS
The proposed strategy relies on the suppression of a chromosomal amber mutation by a plasmid-borne function. We first introduced a nonsense mutation into the essential Escherichia coli thyA gene, resulting in thymidine auxotrophy. The bacterial strain was optimized for the production of small and novel plasmids free of antibiotic resistance markers (pFARs) and encoding an amber suppressor t-RNA. Finally, the potentiality of pFARs as eukaryotic expression vectors was assessed by monitoring luciferase activities after electrotransfer of LUC-encoding plasmids into various tissues.


RESULTS
The introduction of pFARs into the optimized bacterial strain restored normal growth to the auxotrophic mutant and allowed an efficient production of monomeric supercoiled plasmids. The electrotransfer of LUC-encoding pFAR into muscle led to high luciferase activities, demonstrating an efficient gene delivery. In transplanted tumours, transgene expression levels were superior after electrotransfer of the pFAR derivative compared to a plasmid carrying a kanamycin resistance gene. Finally, in skin, whereas luciferase activities decreased within 3 weeks after intradermal electrotransfer of a conventional expression vector, sustained luciferase expression was observed with the pFAR plasmid.


CONCLUSIONS
Thus, we have designed a novel strategy for the efficient production of biosafe plasmids and demonstrated their potentiality for nonviral gene delivery and high-level transgene expression in several tissues.
Marie
Corinne
C
Université Paris Descartes, Faculté de Pharmacie, Unité de Pharmacologie Chimique et Génétique et d'Imagerie, Ecole Nationale Supérieure de Chimie de Paris, INSERM U1022, CNRS UMR8151, Paris, France. corinne.marie@univ-paris5.fr
Vandermeulen
Gaëlle
G
Quiviger
Mickaël
M
Richard
Magali
M
Préat
Véronique
V
Scherman
Daniel
D
D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D045744:Cell Line, Tumor; D017931:DNA Primers / Q000235:genetics; D004352:Drug Resistance, Microbial / Q000235:genetics; D018274:Electroporation; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005260:Female; D016153:Genes, Suppressor; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism*; D008156:Luciferases; D051379:Mice; D008807:Mice, Inbred BALB C; D010957:Plasmids / Q000235:genetics / Q000378:metabolism*; D013940:Thymidylate Synthase / Q000235:genetics
D015415:Biomarkers; D017931:DNA Primers; D008156:Luciferases; D013940:Thymidylate Synthase