1. J Gene Med. 2010 Apr;12(4):323-32.

pFARs, plasmids free of antibiotic resistance markers, display high-level
transgene expression in muscle, skin and tumour cells.

Marie C, Vandermeulen G, Quiviger M, Richard M, Préat V, Scherman D.

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

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.

PMID: 20209487 [PubMed - in process]