The eye is an excellent candidate for gene therapy as it is immune privileged and much of the disease-causing genetics are well understood. Towards this goal, we evaluated the efficiency of compacted DNA nanoparticles as a system for non-viral gene transfer to ocular tissues. The compacted DNA nanoparticles examined here have been shown to be safe and effective in a human clinical trial, have no theoretical limitation on plasmid size, do not provoke immune responses, and can be highly concentrated. Here we show that these nanoparticles can be targeted to different tissues within the eye by varying the site of injection. Almost all cell types of the eye were capable of transfection by the nanoparticle and produced robust levels of gene expression that were dose-dependent. Most impressively, subretinal delivery of these nanoparticles transfected nearly all of the photoreceptor population and produced expression levels almost equal to that of rod opsin, the highest expressed gene in the retina. As no deleterious effects on retinal function were observed, this treatment strategy appears to be clinically viable and provides a highly efficient non-viral technology to safely deliver and express nucleic acids in the retina and other ocular tissues.

RPE65 is an isomerohydrolase expressed in retinal pigment epithelium. It is critical for the regeneration of the visual pigment necessary for both rod and cone-mediated vision. Mutations in human RPE65 cause Leber's congenital amaurosis and other forms of autosomal recessive retinitis pigmentosa which are associated with early-onset blindness. Several RPE65 animal models including two different mouse models and a naturally occurring canine model have been thoroughly characterized to determine the mechanisms that underlie RPE65 associated retinal dystrophies. More recently, substantial effort has gone into designing gene therapies for these diseases. Based on several encouraging reports from animal models, at least three clinical trials are currently underway for the treatment of LCA using modified AAV vectors carrying the RPE65 cDNA and have reported positive preliminary results.

Mutations in the photoreceptor-specific flippase ABCA4 are associated with Stargardt disease and many other forms of retinal degeneration that currently lack curative therapies. Gene replacement is a logical strategy for ABCA4-associated disease, particularly given the current success of traditional viral-mediated gene delivery, such as with adeno-associated viral (AAV) vectors. However, the large size of the ABCA4 cDNA (6.8 kbp) has hampered progress in the development of genetic treatments. Nonviral DNA nanoparticles (NPs) can accommodate large genes, unlike traditional viral vectors, which have capacity limitations. We utilized an optimized DNA NP technology to subretinally deliver ABCA4 to Abca4-deficient mice. We detected persistent ABCA4 transgene expression for up to 8 months after injection and found marked correction of functional and structural Stargardt phenotypes, such as improved recovery of dark adaptation and reduced lipofuscin granules. These data suggest that DNA NPs may be an excellent, clinically relevant gene delivery approach for genes too large for traditional viral vectors.

The purpose of the present study was to test the therapeutic efficiency and safety of compacted‐DNA nanoparticle‐mediated gene delivery into the subretinal space of a juvenile mouse model of retinitis pigmentosa. Nanoparticles containing the mouse opsin promoter and wild‐type mouse Rds gene were injected subretinally into mice carrying a haploinsufficiency mutation in the retinal degeneration slow ( rds ^+/− ) gene at postnatal day (P)5 and 22. Control mice were either injected with saline, injected with uncompacted naked plasmid DNA carrying the Rds gene, or remained untreated. Rds mRNA levels peaked at postinjection day 2 to 7 (PI‐2 to PI‐7) for P5 injections, stabilized at levels 2‐fold higher than in uninjected controls for both P5 and P22 injections, and remained elevated at the latest time point examined (PI‐120). Rod function (measured by electroretinography) showed modest but statistically significant improvement compared with controls after both P5 and P22 injections. Cone function in nanoparticle‐injected eyes reached wild‐type levels for both ages of injections, indicating full prevention of cone degeneration. Ultrastructural examination at PI‐120 revealed significant improvement in outer segment structures in P5 nanoparticle‐injected eyes, while P22 injection had a modest structural improvement. There was no evidence of macrophage activation or induction of IL‐6 or TNF‐α mRNA in P5 or P22 nanoparticle‐dosed eyes at either PI‐2 or PI‐30. Thus, compacted‐DNA nanoparticles can efficiently and safely drive gene expression in both mitotic and postmitotic photoreceptors and retard degeneration in this model. These findings, using a clinically relevant treatment paradigm, illustrate the potential for application of nanoparticle‐based gene replacement therapy for treatment of human retinal degenerations.—Cai, X., Conley, S. M., Nash, Z., Fliesler, S. J., Cooper, M. J., Naash, M. I. Gene delivery to mitotic and postmitotic photoreceptors via compacted DNA nanoparticles results in improved phenotype in a mouse model of retinitis pigmentosa. FASEB J. 24, 1178–1191 (2010). www.fasebj.org