Session Speaker

Compacted DNA Nanoparticles, a Non-viral Gene Delivery for Ocular Diseases
Muna I. Naash
USA

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. Expression plasmids (with either eGFP or photoreceptor-specific genes) transcriptionally-controlled by different promoters were compacted into neutral-charged DNA nanoparticles (having a diameter < 8 nm) using polyethylene glycol-substituted lysine peptides and injected subretinally and in some cases intravitreally into the mouse eyes of wild-type (BALB/c) or retinal degeneration models.

The goal is to target photoreceptors and retinal pigment epithelial (RPE) cells. In a set of control animals, either saline or naked plasmid DNA was injected at a volume and a concentration as the nanoparticles. At different times post-injection, eyes were evaluated functionally, structurally and biochemically. Shortly after injections, transgene expression was evaluated by real time RT-PCR and immunehistochemistry. Immunohisto-chemistry demonstrated the ability of these nanoparticles to transfect and express the transgene in vivo at extraordinarily high efficiencies and as early as 2 days and as late as 10 months post-injection. After subretinal injection, eGFP or other photoreceptor-specific genes were detected in almost 100% of the photoreceptors when photoreceptor-specific promoters are used as well as in the inner nuclear layer, RPE, and optic nerve when CMV or Chicken-beta-actin promoters are used. By qRT-PCR, 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 or other transgenes 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 have been underway utilizing this technology to rescue several well characterized animal models of retinal disease.