21 research outputs found
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
Single-stranded oligonucleotide-mediated in vivo gene repair in the rd1 retina
PURPOSE: The aim of this study was to test whether oligonucleotide-targeted gene repair can correct the point mutation in genomic DNA of PDE6b(rd1) (rd1) mouse retinas in vivo.
METHODS: Oligonucleotides (ODNs) of 25 nucleotide length and complementary to genomic sequence subsuming the rd1 point mutation in the gene encoding the beta-subunit of rod photoreceptor cGMP-phosphodiesterase (beta-PDE), were synthesized with a wild type nucleotide base at the rd1 point mutation position. Control ODNs contained the same nucleotide bases as the wild type ODNs but with varying degrees of sequence mismatch. We previously developed a repeatable and relatively non-invasive technique to enhance ODN delivery to photoreceptor nuclei using transpalpebral iontophoresis prior to intravitreal ODN injection. Three such treatments were performed on C3H/henJ (rd1) mouse pups before postnatal day (PN) 9. Treatment outcomes were evaluated at PN28 or PN33, when retinal degeneration was nearly complete in the untreated rd1 mice. The effect of treatment on photoreceptor survival was evaluated by counting the number of nuclei of photoreceptor cells and by assessing rhodopsin immunohistochemistry on flat-mount retinas and sections. Gene repair in the retina was quantified by allele-specific real time PCR and by detection of beta-PDE-immunoreactive photoreceptors. Confirmatory experiments were conducted using independent rd1 colonies in separate laboratories. These experiments had an additional negative control ODN that contained the rd1 mutant nucleotide base at the rd1 point mutation site such that the sole difference between treatment with wild type and control ODN was the single base at the rd1 point mutation site.
RESULTS: Iontophoresis enhanced the penetration of intravitreally injected ODNs in all retinal layers. Using this delivery technique, significant survival of photoreceptors was observed in retinas from eyes treated with wild type ODNs but not control ODNs as demonstrated by cell counting and rhodopsin immunoreactivity at PN28. Beta-PDE immunoreactivity was present in retinas from eyes treated with wild type ODN but not from those treated with control ODNs. Gene correction demonstrated by allele-specific real time PCR and by counts of beta-PDE-immunoreactive cells was estimated at 0.2%. Independent confirmatory experiments showed that retinas from eyes treated with wild type ODN contained many more rhodopsin immunoreactive cells compared to retinas treated with control (rd1 sequence) ODN, even when harvested at PN33.
CONCLUSIONS: Short ODNs can be delivered with repeatable efficiency to mouse photoreceptor cells in vivo using a combination of intravitreal injection and iontophoresis. Delivery of therapeutic ODNs to rd1 mouse eyes resulted in genomic DNA conversion from mutant to wild type sequence, low but observable beta-PDE immunoreactivity, and preservation of rhodopsin immunopositive cells in the outer nuclear layer, suggesting that ODN-directed gene repair occurred and preserved rod photoreceptor cells. Effects were not seen in eyes treated with buffer or with ODNs having the rd1 mutant sequence, a definitive control for this therapeutic approach. Importantly, critical experiments were confirmed in two laboratories by several different researchers using independent mouse colonies and ODN preparations from separate sources. These findings suggest that targeted gene repair can be achieved in the retina following enhanced ODN delivery
Traitement du kératocône par anneaux intracornéens Intacs® implantés par chirurgie assistée du laser femtoseconde IntraLase®
PARIS7-Xavier Bichat (751182101) / SudocPARIS-BIUM (751062103) / SudocSudocFranceF
Nouvelles stratégies thérapeutiques dans les dégénérescences rétiniennes héréditaires (correction génique ciblée et apport de facteur neurotrophique par vecteurs non viraux)
Les dystrophies rétiniennes héréditaires regroupent un ensemble de maladies dégénératives de la rétine conduisant progressivement à une cécité. Aucun traitement n'est à ce jour disponible. La thérapie génique par vecteurs viraux a apporté la preuve que la fonction visuelle peut être restituée quand l'anomalie génétique est compensée, mais les obstacles à l'utilisation de virus pour une maladie qui ne menace pas le pronostic vital limitent les applications cliniques. Ce travail s'intègre dans celui d'une équipe entièrement dédiée au développement de thérapies oculaires innovantes par vecteurs non viraux. Mon travail porte sur l'évaluation de deux stratégies thérapeutiques complémentaires appliquées à la souris rd1, porteuse d'une mutation ponctuelle dans la sous-unité ß du gène de la phosphodiesterase, responsable d'une dégénérescence post-natale des photorécepteurs en 4 semaines. L'une des stratégies tente de "corriger" la mutation ponctuelle par l'administration d'oligonucléotides portant la séquence ADN native du gène. Les oligonucléotides sont injectés dans le vitré et leur pénétration dans les photorécepteurs est induite par l'application d'un courant iontophorèse transpalpébrale. L'autre stratégie, applicable à plus court terme, vise à améliorer la biodisponibilité du GDNF (Glial Derived Neurotrophic Factor) en induisant sa libération prolongée dans le vitré par son encapsulation dans les microsphères polymériques biodégradables de PLGA. La distribution d'un oligonucléotide a été évaluée sur coupe par histochimie. L'efficacité de ces thérapeutiques est évaluée à 28 jours, quand la quasi totalité des bâtonnets (qui représentent 90 % des photorécepteurs chez la souris) a dégénéré. Le dénombrement des photorécepteurs survivants est réalisé sur coupe, et l'identification des bâtonnets est réalisée par le marquage de la rhodopsine, protéine spécifique des bâtonnets par immunohistochimie. Nos résultats ont montré : - Que l'iontophorèse transpalpébrale réalisée dans des conditions que j'ai définies (ionphorèse anionique, courant 1,5 mA, pendant 5 minutes, réalisée avant l'injection intravitréenne), augmente significativement la transfection des oligonucléotides dans les noyaux des photorecepteurs. - Q'un oligonucléotide spécifique favorise significativement la survie des photorécepteurs à 28 jours et induit la synthèse de la phosphodiestérase dans un nombre réduit de cellules, ce qui est un argument phenotypique d'une possible correction génique. - Que le GDNF encapsulé dans des microsphères polymériques, administrées par voie intravitréenne, favorise significativement la survie des photorécepteurs à 28 jours. Cette survie des supérieure à celle observée par d'autres équipes avec des injections répétées sous-rétiniennes de GDNF. Des travaux complémentaires doivent être réalisés afin de vérifier que les effets histologiques observés sont corrélés à une activité électrorétinographique. Grâce à l'utilisation d'un courant électrique favorisant la transfection des photorécepteurs, nous apportons la première démonstration que la correction génique ciblée est réalisable dans des cellules neuronales post-mitotiques. Son efficacité est encore faible mais pourrait bénéficier d'améliorations techniques et des effets synergiques pourraient résulter d'une combinaison avec les microsphères de GDNF. Ces résultats sont encourageants et ouvrent de nouvelles perspectives thérapeutiques dans le traitement des maladies cécitantes.ST QUENTIN EN YVELINES-BU (782972101) / SudocPARIS-BIUM (751062103) / SudocSudocFranceF
Severe Keratitis Caused by Pseudomonas aeruginosa Successfully Treated with Ceftazidime Associated with Acetazolamide
Purpose. To report a case of microbial keratitis caused by Pseudomonas aeruginosa treated with a combination of acetazolamide and ceftazidime. Methods. Case report. Results. We report the case of a 17-year-old contact lens-wearing female who developed severe keratitis due to Pseudomonas aeruginosa temporarily healed with topical fortified antibiotic eye drops. After few days, the patient relapsed, and topical and intravenous ceftazidime were added. Concomitantly, oral administration of acetazolamide was prescribed. This carbonic anhydrase inhibitor was added to the antibiotic regimen in order to decrease the anterior chamber pH, and then, the ceftazidime ionization. By lowering the state of ionization of the antibiotic in the aqueous humor, its concentration was increased. This was confirmed by an improvement of the patient within few days and a rapid eradication of the infection. Conclusion. This is the first reported case of keratitis caused by P. aeruginosa successfully treated using acetazolamide as an enhancer of ceftazidime effectiveness
Learning scenarios for a 3D virtual environment: The case of special relativity.
International audienceSpecial Relativity, as introduced by Einstein, is regarded as one of the most important revolutions in the history of physics. Nevertheless, the observation of direct outcomes of this theory on mundane objects is impossible because they can only be witnessed when travelling at relative speeds approaching the speed of light c. These effects are so counterintuitive and contradicting with our daily understanding of space and time that physics students find it hard to learn special relativity beyond mathematical equations and to understand the deep implications of the theory. Although we cannot travel at the speed of light, Virtual Reality (VR) makes it possible to experiment the effects of relativity in a 3D immersive environment (a CAVE: Cave Automatic Virtual Environment). The use of the immersive environment is underpinned by the development of dedicated learning scenarios created through a dialectic between VR-related computational constraints and cognitive constraints that include students’ difficulties
Learning scenario for a 3D virtual environment: the case of Special Relativity
International audienceSpecial Relativity, as introduced by Einstein, is regarded as one of the most important revolutions in the history of physics. Nevertheless, the observation of direct outcomes of this theory on mundane objects is impossible because they can only be witnessed when travelling at relative speeds approaching the speed of light c. These effects are so counterintuitive and contradicting with our daily understanding of space and time that physics students find it hard to learn special relativity beyond mathematical equations and to understand the deep implications of the theory. Although we cannot travel at the speed of light, Virtual Reality (VR) makes it possible to experiment the effects of relativity in a 3D immersive environment (a CAVE: Cave Automatic Virtual Environment). The use of the immersive environment is underpinned by the development of dedicated learning scenarios created through a dialectic between VR-related computational constraints and cognitive constraints that include students' difficulties
Reasons for stopping Pressurized IntraPeritoneal Aerosol Chemotherapy (PIPAC): A retrospective study to improve future patient selection
Reasons for stopping Pressurized IntraPeritoneal Aerosol Chemotherapy (PIPAC): A retrospective study to improve future patient selection.
To improve the prognosis and maintain quality of life in patients with peritoneal metastasis (PM), a novel treatment has been introduced-pressurized intraperitoneal aerosol chemotherapy (PIPAC). The majority of teams propose at least 3 PIPAC procedures. However, for many patients PIPAC is stopped after only one or two procedures. The aim of this study was to identify the reasons for stopping PIPAC after only one or two procedures and to establish a profile of poor candidates. This retrospective, multicenter cohort study included all patients who underwent PIPAC in three French expert centers between 2015 and 2021. A total of 268 PIPAC procedures were performed in 89 patients. Of them, 48.3% of patients underwent fewer than three procedures: 28.1% had one, 20.2% two and 51.7% three or more PIPAC procedures. The main reason for stopping PIPAC, regardless of the number of procedures, was disease progression, in 55.8% of cases. Other reasons for stopping PIPAC were non-access to the abdominal cavity (7.9%), conversion to cytoreductive surgery (13.5%), post-PIPAC adverse events (7.9%), patients' wishes (10.1%) and death (2.2%). In univariate analysis, patients who received fewer than three PIPACs less frequently had chemotherapy beforehand (91% vs 100%, p = 0.05), less frequently had bimodal treatment (70% vs 87%, p = 0.04), had more ascites (median 80 ml vs 50 ml, p = 0.05) and more frequently had carcinomatosic ascites (48.8% vs 23.9%, p < 0.01). Performing PIPAC alone in chemotherapy-naïve patients with ascites should be avoided