Novel drug delivery systems targeting intraocular tissues

The availability of new therapeutic molecules for the management of chronic intraocular diseases has highlighted the deficiency of drug delivery systems for their administration. New research programs on drug delivery methods designed to reduce the administration frequency have led to the development of various polymers, biodegradable or not, that release therapeutic molecules directly into the vitreous cavity. Two innovating ocular delivery methods are now available, based on the use of electrical current: iontophoresis, a non-invasive intraocular delivery method for various molecules, and plasmid electroporation into the ciliary muscle, in which the muscle produces a therapeutic molecule directly inside the posterior chamber for several months. In the near future, we will be able to administer therapeutic molecules to treat a specific disease using a method of administration targeting a specific intraocular tissue.La mise sur le marché de molécules innovantes pour le traitement des maladies oculaires chroniques a révélé la pauvreté des moyens de les administrer, mais elle a aussi ouvert la voie à la recherche dans ce domaine. Dans le but de limiter la fréquence des administrations, des polymères, biodégradables ou non, ont été développés pour libérer des principes actifs directement dans la cavité vitréenne. L'utilisation du courant électrique est à l'origine de deux techniques d'administration innovantes: l'iontophorèse, qui permet d'administrer des principes actifs en intraoculaire sans aucune effraction, et l'électroporation de plasmide dans le muscle ciliaire, qui permet de faire produire par ce muscle une molécule thérapeutique, directement dans la chambre postérieure, et ce pendant plusieurs mois. Dans le futur proche, pour chaque maladie nécessitant un principe actif particulier, nous disposerons d'une méthode adaptée d'administration ciblant spécifiquement un tissu oculaire

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