Abstract
Purpose :
Gene therapy has become a promising tool for the treatment of inherited retinal diseases (IRDs) and gene transfer to ocular tissue is typically mediated through the intraocular injection of recombinant vectors derived from mammalian viruses, the most common being adeno-associated virus (rAAV). While rAAVs have demonstrated the ability to transduce a variety of ocular cell types efficiently, rAAV gene therapies have limitations, including a limited coding capacity, high cost, and immunogenicity. Gene transfer can also be mediated through vectors of non-viral origin (liposomes, polyplexes, and nanoparticles); however, these vectors also have limitations: short-term expression, immunogenicity, and low transfection efficiencies. Lipid Nanoparticles (LNPs) are the most clinically advanced non-viral vector, reporting robust transfection efficiencies of RNA in the liver, lung and eye. Given that large gene delivery with a non-viral vector would greatly benefit the IRD field, we utilized a modified LNP (LNPx) to deliver dsDNA to the retina aiming to facilitate long-term gene expression.
Methods :
We generated LNPx encapsulating GFP mRNA or mCherry dsDNA. These LNPs were administered individually and in combination via subretinal injection (1μL containing 100ng/eye) in wild-type (C57BL/6J) mice (N=3 eyes/group). An equal volume of buffer was delivered to a fourth group as a control. Gene expression was followed via confocal SLO at 1-, 3-, 7-, 14-, and 21-days post-injection. At 21 days post-injection, mice also underwent optical coherence tomography (OCT) imaging to rule out structural damage to the retina. Following final cSLO and OCT assessments, mice were euthanized, and their eyes were harvested for histological sections.
Results :
cSLO showed successful gene expression of both mRNA and dsDNA containing LNPs. mRNA expression begins as early 3 days post-injection and diminishes by 14 days. dsDNA expression began around 7 days post injection and continues through 14 days. OCT showed no obvious retinal thinning but did indicate of inflammatory cells in all groups. Histology is ongoing.
Conclusions :
While gene therapy remains a promising tool for the treatment of IRDs, non-viral, large gene delivery resulting in long-term expression remains a major bottleneck. Herein, we show that the non-viral LNP vector is capable of delivering dsDNA to retinal cells, resulting in long-term gene expression, a vital steppingstone for large gene delivery.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.