Abstract
Purpose :
Increased oxidative stress is important in the pathophysiology of multiple optic neuropathies, leading to retinal ganglion cell (RGC) apoptosis and visual impairment. In vivo studies show that SIRT1, a NAD+-dependent deacetylase, reduces RGC loss in several optic neuropathy disease models. The current study uses an in vitro model of oxidative stress to investigate neuroprotective effects of a SIRT1 gene therapy in human RGCs differentiated from induced-pluripotent stem cells (iPSC-RGCs).
Methods :
RGCs were differentiated from iPSCs derived from a healthy human donor. RGCs were cultured and matured for 40 days, with cellular identity confirmed using RT-qPCR and immunocytochemistry. iPSC-RGCs were transduced with 1E3 vector genomes/cell of an adeno-associated virus, serotype 2 (AAV2) vector expressing SIRT1 under the RGC-selective promoter, gamma-synuclein, while expression of eGFP was used as a control. Cells transduced with AAV2 SIRT1/eGFP were plated at a density of 2E4 cells/well for 3 repeat experiments. Tert-butyl hydroperoxide (tbHO) was used to induce reactive oxygen species. Transduced iPSC-RGCs were exposed to varying concentrations of tbHO for 24 hr. RGC viability was determined by MTT assay. Survival was reported for the tbHO concentration that produced the greatest difference in percent viability between SIRT1 versus eGFP groups in each experiment.
Results :
The ability of increasing concentrations of tbHO to decrease cell viability was confirmed in non-transduced iPSC-RGCs. Percent viability was 33.86% at 350mM, 39.01% at 300mM, 62.17% at 250mM, 68.46% at 200mM, 72.98% at 150mM, 90.40% at 100mM, and 94.87% at 50mM. A similar dose-response curve was repeated for each experiment. Percent viability was significantly higher in SIRT1-treated iPSC-RGCs (93.15±4.97%) as compared to GFP-treated iPSC-RGCs (76.93±5.47%) exposed to the optimal tbHO concentration (mean±SEM; N=3 experiments). This difference in percent viability was significant in each of the three experiments: 11.83% (P=.03), 18.04% (P=.002), and 18.81% (P=.04), with greater survival in SIRT1-treated iPSC-RGCs.
Conclusions :
Results demonstrate that SIRT1 gene therapy increases survival of human iPSC-RGCs exposed to tbHO. The data corroborates previously reported findings of the protective effects of SIRT1 in animal models of optic neuropathy. This approach may be useful in determining patient-specific responses to SIRT1 gene therapy using iPSC-RGCs.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.