M. Doat

Plasmid electrotransfer of eye ciliary muscle: principles and therapeutic efficacy using hTNF-α soluble receptor in uveitis

Due to its small size and particular isolating barriers, the eye is an ideal target for local therapy. Recombinant protein ocular delivery requires invasive and painful repeated injections. Alternatively, a transfected tissue might be used as a local producer of transgene‐encoded therapeutic protein. We have developed a nondamaging electrically mediated plasmid delivery technique (electrotransfer) targeted to the ciliary muscle, which is used as a reservoir tissue for the long‐lasting expression and secretion of therapeutic proteins. High and long‐lasting reporter gene expression was observed, which was restricted to the ciliary muscle. Chimeric TNF‐α soluble receptor (hTNFR‐Is) electrotransfer led to elevated protein secretion in aqueous humor and to drastic inhibition of clinical and histological inflammation scores in rats with endotoxininduced uveitis. No hTNFR‐Is was detected in the serum, demonstrating the local delivery of proteins using this method. Plasmid electrotransfer to the ciliary muscle, as performed in this study, did not induce any ocular pathology or structural damage. Local and sustained therapeutic protein production through ciliary muscle electrotransfer is a promising alternative to repeated intraocular protein administration for a large number of inflammatory, degenerative, or angiogenic diseases.

Stable transmission of targeted gene modification using single-stranded oligonucleotides with flanking LNAs

Targeted mutagenesis directed by oligonucleotides (ONs) is a promising method for manipulating the genome in higher eukaryotes. In this study, we have compared gene editing by different ONs on two new target sequences, the eBFP and the rd1 mutant photoreceptor βPDE cDNAs, which were integrated as single copy transgenes at the same genomic site in 293T cells. Interestingly, antisense ONs were superior to sense ONs for one target only, showing that target sequence can by itself impart strand-bias in gene editing. The most efficient ONs were short 25 nt ONs with flanking locked nucleic acids (LNAs), a chemistry that had only been tested for targeted nucleotide mutagenesis in yeast, and 25 nt ONs with phosphorothioate linkages. We showed that LNA-modified ONs mediate dose-dependent target modification and analyzed the importance of LNA position and content. Importantly, when using ONs with flanking LNAs, targeted gene modification was stably transmitted during cell division, which allowed reliable cloning of modified cells, a feature essential for further applications in functional genomics and gene therapy. Finally, we showed that ONs with flanking LNAs aimed at correcting the rd1 stop mutation could promote survival of photoreceptors in retinas of rd1 mutant mice, suggesting that they are also active in vivo.

380 Toxicité endothéliale cornéenne du peroxynitrite dans les stress oxydatifs

Introduction Nous avons precedemment montre la presence de iNOS et l’impact du stress nitrosant sur la couche de cellules endotheliales (CE) au cours du rejet aigu de greffe sur modele animal (1). Le peroxynitrite (ONOO-) pourrait contribuer a la toxicite CE. Nous avons donc explore l’effet de l’injection en chambre anterieure (CA) in vivo chez le rat a diverses concentrations de ONOO- et observe les consequences sur les CE. Objectifs et Methodes Une courbe de decomposition a temperature ambiante du ONOO- a ete realisee pour chaque solution experimentale. 10 μl d’une des solutions suivantes ont ete injectes en CA de rat Lewis (n = 18 ; 3 yeux/solution, 1 œil par rat) : 500 μm, 250 μm, ou 50 μm ONOO- in 0,05 m NaOH (excipient), ONOO- denature (dONOO), excipient seul ou NaCl 0.9 %. La densite cellulaire endotheliale (DCE) et la pachymetrie ont ete mesurees en microscopie confocale in vivo (HRTII, Heidelberg Engineering, Allemagne). Resultats A 6 heures post injection, la pachymetrie moyenne (Pm) etait respectivement de 158 ± 16 μm, 136 ± 8 μm, 198 ± 9 μm, 192 ± 14 μm 234 ± 13 μm, 306 ± 33 μm et 272 ± 20 μm pour les groupes : non injecte, NaCl ; excipient, dONOO ; 50, 250 ou 500 μm de ONOO-. La Pm augmentait pour le groupe excipient compare au non injecte (p = 0.01) mais l’augmentation etait tres significative comparee aux groupes 250 et 500 μm de ONOO- (p Discussion Le ONOO est produit en cas de stress oxydatif local. Sa toxicite proper sur les cellules corneennes n’avait pas encore ete demontree en conditions in vivo. Conclusion Le peroxynitrite injecte en CA et capable d’induire par lui-meme une perte cellulaire et fonctionnelle endotheliale, y compris a faible concentration.