Design and Evaluation of Targeted mRNA Delivery Systems to the Retina

Abstract

Glaucoma is a neurodegenerative eye disease traditionally linked to elevated intraocular pressure (IOP). However, recent studies have revealed a complex pathogenic process involving the interplay of multiple factors, leading to the death of retinal ganglion cells (RGCs) and ultimately resulting in vision loss. Current treatments primarily aim to normalize IOP levels. Nonetheless, they are limited by issues and have often failed to prevent eventual vision loss. Neuroprotective gene therapy presents a promising approach to safeguarding RGCs and promoting their survival against potential damage from elevated IOP. This can be achieved by increasing neurotrophic factors (NTFs) and reducing the activation of apoptotic pathways. In this project, the main objective was to develop an effective non-viral lipid nanoparticle (LNP) system for mRNA for intravitreal (IVT) delivery in the eye using a second-generation parent 18-7NH-18 gemini surfactant (GS) and three derived cell-adhesive-peptide (CAP p1, p3 and p5) conjugated GSs: 18-7Np1-18, 18-7Np3-18, and 18-7Np5-18, along with other lipids. The goal was to enable effective mRNA delivery to the retina as an initial step towards developing a non-viral neuroprotective gene therapy for glaucoma. Gem++ION hybrid lipid nanoparticle (LNP) system was developed and prepared using Ultrasonication Method 1-3h (USM1-3). The parent Gem++ION Supra formulation and two peptide-conjugated Gem++ION (pGem++ION) Supra formulations were evaluated in vitro in two retinal cell lines and in vivo in CD-1 mouse model. The results demonstrated >90% transfection efficiency (TE) in vitro for all three formulations in both cell lines. In the in vivo study, the two pGem++ION Supra formulations, after 48 hours of intravitreal administration, showed GFP expression in various retinal layers, consistent with the Cy5-labelled LNP biodistribution results by confocal microscopy. Additionally, the three Gem++ION Supra LNP formulations exhibited GFP expression in the anterior eye segment, including the ciliary body, iris, and cornea. Overall, in this project, a novel hybrid dicationic+ionizable lipid LNP system with CAP targeting ligands, termed Gem++ION Supra, and pGem++ION Supra formulations, with high efficiency to deliver and transfect retinal cells, particularly the RGCs, both in vitro and in vivo, were developed, paving the way for further development in neuroprotective gene therapy for glaucoma.