Abstract
BackgroundThe 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.Methods and FindingsHere 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.ConclusionsAs 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.
Highlights
The eye is comprised of several specialized tissues that work together to initiate visual perception in response to photons of light
Amplification was only detected in positive control samples of pure nanoparticle DNA, demonstrating sufficient removal of nanoparticle contamination from isolated RNA samples. quantitative RT-PCR (qRT-PCR) was performed in triplicate on each cDNA sample using an iCycler (Bio-Rad Inc.) and DcT values were calculated against the neuronal housekeeping gene hypoxanthine phosphoribosyltransferase (Hprt)
To target different ocular tissues, we varied the site of ocular injection between the intravitreal space and subretinal space as we hypothesized that the area of delivery may cause different cell types to express enhanced green fluorescent protein (EGFP)
Summary
The eye is comprised of several specialized tissues that work together to initiate visual perception in response to photons of light Any insult to these tissues results in a consequence to vision and an impact on the quality of life for the patient. 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. 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
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