668. Non-Viral Gene Delivery for Ocular Diseases with Compacted DNA Nanoparticles

Top of pageAbstract Our objective is to develop an effective and robust therapeutic vector system which can be utilized in the treatment of genetically-based blinding diseases. In the present study we have evaluated the ability of compacted DNA nanoparticles to transfect mouse ocular tissues in vivo. EGFP expression plasmids transcriptionally-controlled by the CMV promoter were compacted into neutral-charged DNA nanoparticles (having a diameter < 15 nm) using polyethylene glycol-substituted lysine peptides and injected subretinally or intravitreally into the eyes of adult BALB/c mice. Both delivery routes were examined in order to target photoreceptors, RPE, and optic nerve cells (subretinal) or inner retinal cells (intravitreal). In a set of control animals, naked plasmid DNA was injected at the same concentration as the nanoparticles or a mock injection was performed. After 2 days post-injection, mice were euthanized and the retinas were assayed for EGFP expression by quantitative real-time RT-PCR (qRT-PCR) and immunohistochemistry. Significant levels of EGFP expression were obtained by either subretinal or intravitreal delivery of the compacted DNA nanoparticles. Immunohistochemistry demonstrated the ability of these nanoparticles to transfect and express EGFP in vivo at extraordinarily high efficiencies. After subretinal injection, EGFP was detected in almost 100% of the photoreceptors and abundant expression was observed in the inner nuclear layer, RPE, and optic nerve. By qRT-PCR, EGFP mRNA levels were comparable in abundance to rhodopsin mRNA. Mice injected intravitreally showed very efficient EGFP expression in the cells of the inner retina, including the ganglion cell layer. After either subretinal or intravitreal delivery, minimal or no expression of EGFP was detected with naked plasmid or mock-injected controls. Ocular delivery of the DNA nanoparticles did not induce any apparent toxicity. Compacted DNA nanoparticles can efficiently target post-mitotic cells of the retina and yield high levels of transgene expression. Transfection of different retinal cell layers can be achieved by the route of delivery. This non-viral system is a safe and very effective tool for ocular gene therapy. Further studies are underway utilizing this technology to rescue several well characterized animal models of retinal disease.