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Renee Christine Ryals, Milan Gautam, Jonathan Stoddard, Rene Reynaga, Scott Shubert, Lauren Renner, Jeonghwan Kim, Wayne Tschetter, Ian Fries, Andreas Lauer, Martha Neuringer, Gaurav Sahay; Lipid nanoparticles transfect multiple retinal cell types in the non-human primate.. Invest. Ophthalmol. Vis. Sci. 2022;63(7):3475.
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© ARVO (1962-2015); The Authors (2016-present)
Our lab has generated novel lipid nanoparticles (LNPs) for delivery of gene editors to the retina and demonstrated robust transfection of the rodent retina. The goal of this study was to evaluate retinal transfection efficiency in a more clinically relevant model, the non-human primate (NHP).
Novel LNP variants were prepared via rapid microfluidic mixing of an organic phase containing the lipids (ionizable lipid, DSPC, sterols, and PEG) and aqueous phase containing GFP mRNA. LNPs were characterized for hydrodynamic radius, polydispersity index (PDI), encapsulation efficiency and zeta potential (ZP). LNPs were transfected into NHP iPSC-derived RPE cells at multiple doses ranging from 500 ng to 10 µg. Rhesus macaques received baseline optical coherence tomography (OCT), fundus autofluorescence (FAF) and ultra-wide-field imaging. Subretinal surgery was performed by a skilled retinal surgeon using an Alcon Constellation vitrectomy machine to deliver 100 µl (50 µg total) of LNPs to each bleb per eye. No systemic immune suppression was used. At 48 hours post-injection, in vivo retinal imaging was performed and eyes were then harvested for histology and immunohistochemistry (IHC). LNPs were also subretinally delivered to WT C57BL6 mice. In vivo retinal imaging and IHC of retinal sections were performed to characterize intracellular gene expression.
All LNPs had a diameter <85 nm, with a PDI <0.10. The ZP of the particles varied from -1.1 to -5.3 and encapsulation efficiency ranged from 98-99%. GFP expression was observed in NHP iPSC-derived RPE cells at 48 hours post-LNP transfection at all doses tested. Baseline imaging confirmed that all macaques had normal retinal morphology prior to surgery. At 48 hours post-injection, in vivo retinal FAF imaging showed robust GFP expression in the subretinal blebs. IHC demonstrated GFP expression localized to photoreceptors, RPE and Müller glia (Figure 1). Evaluation of the same particles in WT mice on the same day confirmed GFP expression and localization.
LNPs successfully transfected many retinal cells in the NHP including photoreceptors, RPE and Müller glia post-subretinal delivery. These data demonstrate the translatability of LNP-mediated gene delivery to retinal cells across species and support their development as delivery systems for retinal disease therapies.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.
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