Abstract
Purpose :
Although lipid nanoparticles (LNPs) effectively deliver genes to the retina, most of the gene expression is still limited to the retinal pigmented epithelium (RPE) and Müller glia. This study tested the hypothesis that modifications to LNPs can steer LNP delivery of mRNA to the photoreceptors. Successful gene delivery to RPE, Müller glia, and photoreceptors expands the utility of LNPs for treating inherited retinal degenerations.
Methods :
LNP variants were prepared via rapid microfluidic mixing of an organic phase containing lipids and an aqueous phase containing mRNA. The organic phase, consisting of ionizable lipid, DSPC, sterols, DMG-PEG, and PEGs at different molar ratios, was rapidly mixed with the aqueous phase having Cre or mCherry mRNA in the citrate buffer pH 4.0. LNPs were characterized for hydrodynamic radius and polydispersity index (PDI) using dynamic light scattering (DLS) and mRNA encapsulation efficiency using a modified Quant-iT RiboGreen RNA reagent. First, LNPs were evaluated for transfection efficiency in multiple cell lines. Then, Cre or mCherry mRNA (300 ng) was delivered via subretinal injections in both Ai9 and NRL-GFP mice. Live in-vivo retinal imaging and immunohistochemistry (IHC) of retinal sections were performed to characterize the intracellular gene expression.
Results :
Novel LNPs had a diameter of < 80 nm with a < 0.13 PDI. The zeta potential (ZP) of the particle varied depending on the functional PEG. The ZP of LNPs without functional PEG was -0.95±0.6. In all cases, encapsulation efficiency was higher than 96% with a spherical shape in morphology. LNP variant had significantly higher transfection efficiencies compared to standard LNPs in photoreceptor cell lines. After delivery of LNP variants in Ai9 mice, robust and selective tdTomato expression was observed using fundus imaging. IHC confirmed expression in the photoreceptors and RPE. With the addition of b-sitosterol, expression was observed in Müller glia as well. At 48 h post-injection, mCherry protein expression was localized to the RPE and Müller glia in NRL-GFP mice, further strengthening our observation.
Conclusions :
LNP-based mRNA delivery has largely been restricted to the RPE. We have now demonstrated that LNP variants can deliver genes to the photoreceptors, and the RPE, thus opening the scope for treatments against inherited retinal degenerations.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.