Abstract
Purpose: :
In this study, we evaluated the ability of compacted DNA nanoparticles (NPs) and plasmid DNA to generate efficient and stable transgene expression in mouse retinal pigment epithelial (RPE) cells. Our goal is to develop a non-viral gene delivery system that would be clinically relevant in treating RPE-associated ocular diseases such as Leber congenital Amaurosis (LCA).
Methods: :
We constructed two plasmid vectors containing eGFP and hRPE65 cDNAs driven by human vitelliform macular dystrophy-2 (VMD2) promoter- i) VMD2-eGFP-S/MAR and ii) VMD2-hRPE65-S/MAR. Both vectors were compacted with CK30PEG10K into rod-like DNA NPs (diameter <8-11 nm). NPs or uncompacted DNA (4.3µg/µl) and saline (vehicle) were injected into the subretinal space of 30 day old BALB/c mice or 16 day old RPE65-/- mice. eGFP and hRPE65 expression levels and localization were evaluated by qRT-PCR and immunohistochemistry (IHC). Quantification of eGFP expressing cells was performed on RPE flatmounts to evaluate the distribution of transgene expression. Fundus imaging was performed to show live imaging of DNA distribution as well as eGFP expression. Electroretinography (ERG) was performed to analyze visual rescue and structural maintenance was analyzed by light and electron microscopy (EM) on RPE65-/- mice.
Results: :
qRT-PCR and IHC of retinal whole mounts and cross sections showed high levels of eGFP expression from PI-2 days up to PI-2 years (the longest timepoint tested). ~50-60 % cells throughout the RPE exhibited eGFP expression at 2 years PI. IHC showed eGFP and hRPE65 expression localized specifically to the RPE. At PI-180 days, qRT-PCR for hRPE65 expression showed ~50% of endogenous RPE65 expression for both naked DNA and NP injects. Treated RPE65-/- mice exhibited maintained cone ERGs compared to saline injected cohorts. At PI-180 days, ultrastructural analysis showed fewer or no lipid droplets in the RPE cells of treated mice compared to uninjected controls suggesting reduction in retinyl ester accumulation.
Conclusions: :
Both NPs and naked DNA were able to drive highly efficient and sustained therapeutic gene expression in the RPE. These results suggest that both DNA nanoparticles and naked DNA carrying S/MAR vectors have significant clinical potential for treating RPE-based diseases.
Keywords: gene transfer/gene therapy • retinal pigment epithelium • degenerations/dystrophies