Despite extensive research on many of the genes responsible for inherited retinal degenerations leading to blindness, no effective treatment is currently available for patients affected with these diseases. Among the therapeutic approaches tested on animal models of human retinal degeneration, gene therapy using different types of viral vectors as delivery agents has yielded promising results. We report here our results on a non-invasive, non-viral delivery approach using transscleral iontophoresis for transfer of plasmid DNA into mouse retina. Proof of principle experiments were carried out using plasmid containing GFP cDNA to demonstrate expression of the transferred gene in the retina after single applications of iontophoresis. Various parameters for multiple applications of iontophoresis were optimized to sustain GFP gene expression in mouse photoreceptors. Subsequently, repeated iontophoresis of plasmid containing normal cGMP-phosphodiesterase beta-subunit (beta-PDE) cDNA was performed in the rd1 mouse, an animal model of autosomal recessive retinitis pigmentosa caused by a mutant beta-PDE gene. In normal mice, transscleral iontophoresis of the GFP plasmid provided a significant increase in fluorescence of the retina in the treated versus non-treated eyes. In rd1 mice, repeated iontophoresis of beta-PDE cDNA plasmid partially rescued photoreceptors morphologically, as observed by microscopy, and functionally, as recorded on ERG measurements, without adverse effects. Therefore, transscleral iontophoresis of plasmid DNA containing therapeutic genes may be an efficient, safe and non-invasive method for the treatment of retinal degenerations.

In vitro studies have clearly shown that signaling/guidance proteins can diffuse to their targets. However, it is unclear whether they can travel by diffusion in vivo, or if they are distributed in the tissue by an active mechanism. Retinoschisin, a signaling molecule related to neuropilins, is synthesized and secreted by photoreceptor cells in the outer retina; then it interacts with inner retinal cells contributing to synaptic organization and optic nerve fiber integrity. We developed an assay to examine how retinoschisin, which is secreted a distance away, reaches its inner retinal targets. We found that retinoschisin is preferentially taken up and carried into the inner retina from the retinal outer border (the photoreceptor side) by Müller cells (the main glial cells of the vertebrate retina). This transcytosis is disrupted by DL-alpha-aminoadipic acid, a Müller cell/glia-specific toxin. Our results suggest that glial uptake/transcytosis can provide an effective and precise alternative for distributing signaling molecules in the nervous system.

Usually, photoreceptors interact with other retinal cells through the neurotransmitter glutamate. Here we describe a nonsynaptic interaction via a secreted protein, retinoschisin. Using in situ hybridization, we found that from early postnatal life retinoschisin mRNA is present only in the outer retina of the mouse, and with single-cell RT-PCR we demonstrated its localization in rod and cone photoreceptor cells but not in Müller cells. Western blot analyses of proteins from cultured ocular tissues and microdissected outer and inner retinas, as well as from the culture media of these samples, showed that retinoschisin is secreted from the photoreceptor cells. Immunostaining of permeabilized and nonpermeabilized dissociated retinal cells revealed that retinoschisin is mainly inside and outside the photoreceptors, outside bipolar cells, and associated with plasma membranes of Müller cells and inside their distal processes. Because we showed previously that retinoschisin is distributed all over the retina, our current data suggest that after synthesis and secretion by the photoreceptors, retinoschisin reaches the surface of retinal cells and mediates interactions/adhesion between photoreceptor, bipolar, and Müller cells, contributing to the maintenance of the cytoarchitectural integrity of the retina. These interactions may not occur when the gene encoding retinoschisin is mutated, as it occurs in X-linked juvenile retinoschisis, a disease that results in morphological and electrophysiological defects of the retina.