Abstract
Purpose :
Photoreceptor loss is the final endpoint in nonexudative Age-Related Macular Degeneration (dry-AMD) and no curative treatments are available. Targeted optogene delivery to higher order neurons of retina in geographic atrophy (GA) regions in these patients is a promising approach to improve vision. Here, we report development of an optical coherence tomography (OCT) guided near-infrared laser irradiation platform combined with Surface Plasmon Resonance (SPR) of nanoparticles to deliver ambient-light activatable multi-characteristic opsin II (MCOII) plasmids into human cells and degenerated mouse retina (rd1) in a specially targeted manner. The method is based on nano-enhancement of near-infrared optical field by of bound to specific cells.
Methods :
After pretreatment with functionalized gold nanorods (fGNRs) for 2 hrs, OCT guided laser (980 nm) delivery of MCOII-mCherry plasmids was performed in HEK cells to determine optimal laser parameter and plasmid concentration. Reporter expression was assessed after 48 hrs of laser delivery using confocal fluorescence microscopy. MTT assay was performed to check cytotoxicity after laser gene delivery in HEK cells. For in vivo OCT -guided laser gene delivery, 8-12 week-old rd1 mice were intravitreally injected with a 2µl mixture of fGNR (0.2 µg/µl) and MCOII plasmids (0.5 µg/µl) into each eye. 3-4 hrs post injection, laser delivery was performed in one eye and the contralateral eye was used as a control. The laser beam power, outside eye, was kept at 20 mW for targeted gene delivery. First, real time OCT imaging was used to locate optic nerve, and ~0.7 mm x 0.7 mm rectangular region was irradiated with the scanning laser beam for 400 secs. After 4 weeks of gene delivery, eyes were enucleated, retinal flat mount was prepared and immunostained with anti-mCherry and RBPMS antibody.
Results :
MCOII-mCherry expression in HEK cells was found to vary based on laser beam power. MCOII delivery using optimal laser power (determined in HEK cells) led to mCherry expression in the RGC cells of rd1 mice. MTT assay and OCT measurement after laser delivery indicated minimal cytotoxic effects. Non-targeted regions did not show mCherry expression in either HEK cells or rd1 retina.
Conclusions :
OCT guided laser delivery is an efficient and targeted method to deliver therapeutic genes into degenerated retina.
This is a 2021 ARVO Annual Meeting abstract.