Safe non-viral light-based spatially targeted delivery of therapeutic opsin-encoding genes into specific cell types

Abstract

Purpose : In recent time, gene therapy has proven to be a promising remedial approach for treating visual disorders either by replacement of non functioning gene(s) or by the introduction of light sensitive proteins (opsins) as artificial photoreceptors in retinal cells. Conventional viral vector-based gene delivery method is often confronted with limitations due to packaging size limit, immunogenetic reaction, unintended non-targeted delivery, non-feasibility of repeated re-dosing due to immunorejection, and complicated manufacturing process, leading to significant roadblock in translational success. Most of the non-viral approaches lack spatial and/or cellular specificity and limited by low transfection efficacy and cytotoxicity. In this regard, we have developed an efficient, safe, targeted, light based non-viral gene delivery for ocular therapy.

Methods : We utilized functionalized plasmonic gold nanorods (fGNRs, targeted to attach to specific cell types of retina) and spatially targeted controlled light irradiation for delivering therapeutic genes into mice retina. The contrast in temperature rise in laser-irradiated nanorod-attached cells generate nano-hotspots which allows site-specific delivery of gene into targeted areas. Evaluation of safety and toxicity associated with the transduction of opsin-encoding genes by use of light irradiation was examined by electrophysiology, optical coherence tomography, intraocular pressure and other analytical methods (confocal microscopy, immunohistochemistry).

Results : Targeted in-vivo delivery and expression of opsin-encoding gene in specific retinal cells (e.g. bipolar and ganglion cell layers) was achieved by the use of cell specific fGNRs concurrent with light irradiation. No significant changes in ERG response and IOP was observed upon non-viral transfection in wild type mouse. The immunohistochemistry analysis showed no inflammation or migration of microglia/macrophage due to laser-based gene delivery. In case of blind mice, upon non-viral gene delivery, the functional rescue of light perception was observed.

Conclusions : Based on our results, we conclude that the light based targeted non viral gene delivery method is a safe, effective and translatable approach for targeted delivery of theraputic molecules and will pave the way for successful non-viral gene delivery in future.

This is a 2020 ARVO Annual Meeting abstract.