Résumé :
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Acute unmethylated CG dinucleotide (CpG)-mediated inflammatory response has been shown to be associated with a brief duration of transgene expression in mouse lungs. Indeed, retention of even a single CpG in pDNA was demonstrated to be sufficient to elicit an inflammatory response, whereas CpG-free pDNA vectors did not. Given our interest in muscle gene therapy for myopathies, we wanted to know whether such an observation could be also obtained in the context of in vivo intramuscular transfections. Taking advantage that naked DNA is actually readily able to achieve transgene expression in muscle in vivo, we were able to directly compare the transfection activity of two luciferase encoding plasmids: (1) pTG11033 (Transgene), a CpG-containing pDNA that bears the firefly luciferase cDNA under control of the strong viral CMV promoter; (2) pGEM144 (UK CF trust), entirely CpG-free and that harbours a human elongation factor 1_ promoter upstream to a codon-optimised form of the firefly luciferase. Following animal anaesthesia, naked pDNA (5 ?g in 50 ?L saline or Tyrode) was injected intramuscularly at one site into shaved tibialis anterior muscles of 6-week-old female Swiss mice. Injections were performed using a microfine syringe parallel to the muscle fibers; at least six tibialis anterior muscles were included in each individual set of experiments. Our results showed that, whereas the CpG-containing plasmid allowed only transient transgene expression (<12 days), the CpG-free pDNA achieved a very sustained (>6 months) in vivo intramuscular transgene expression (in the absence of any detectable inflammation). Additionally, mice which received at first a CpG-containing pDNA were then re-injected (when bioluminescence signals were no longer detectable) with either the same plasmid or the CpG-free pDNA. Re-injection of pTG11033 directed a transgene expression peak which however immediately and rapidly decreased (without reaching any stable plateau) and then disappeared few days post-injection. On the contrary, re-injecting pGM144 led to a sustainable luciferase transgene expression. Thus, our results extended the observation made under lung experimental settings showing here that sustainable and recoverable intramuscular transgene expression (in the absence of inflammation) can be achieved with CpG-free nonviral pDNA. In ongoing experiments, synthetic vectors are used to enhance reporter gene expression and tissue-distribution.Travail du rau "DNA-Nanoparticles for Skeletal Muscle and Airway Epithelium In Vivo Gene Therapy" financr l'Association Franse contre les Myopathies
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