Non-viral gene delivery of crystallin gamma S protects retinal cells and increases their proliferation during inflammation

Abstract

Purpose : Previous studies from our laboratory have shown that crystallin gamma S (CryγS) has a protective role against retinal neuroinflammation (Lei et al 2022). Here we developed a non-viral gene therapy with spatiotemporal control of CryγS for use in retinal pathologies to combat neuroinflammation.

Methods : We developed a non-viral gene therapy platform for CryγS delivery with spatiotemporal control of its expression with activation only when required during inflammation. Three different plasmids were constructed: (a) CryγS, (b) CryγS-GFP and (c) GFP alone, all placed under IL1-β promoter. Multifunctional lipids (ECO) were used to transfect human ARPE19 cells. Human ARPE19 cells were transfected with either ECO/pIL1β-CryγS-GFP, ECO/pIL1β-CryγS or ECO/pIL1β-GFP. Immunofluorescence and western blotting were performed to confirm successful transfection. The protective effect of the therapy was evaluated by inducing oxidative stress in control and transfected cells using either hydrogen peroxide (150uM) for 1 hour, LPS (10 ng/ml) or recombinant IL1β (20ng/ml) for 1 day. Cell viability was assessed at 1, 2 and 3 days using alamar blue assay

Results : We achieved 85% transfection efficiency in human ARPE19 cells that received pIL1β-CryγS-GFP nanoparticles and confirmed the results with immunofluorescence and Western blotting. CryγS transfected ARPE19 cells showed resilience against cell death after exposure to hydrogen peroxide (p=0.003, n=4), LPS (p=0.004, n=4), or rIL1β (p=0.001, n=4) and increase in proliferation as compared to control or plasmid alone transfected cells. This was further confirmed by wound healing assay and Ki67 staining, with transfected cells migrating 30% faster over the wound area and having 40% higher Ki67 index as compared to non-transfected cells (p=0.0172, n=3, p=0.0054, n=3 respectively).

Conclusions : In vitro studies support the hypothesis that over-expression of CryγS confers cell protection following exposure to noxious stimuli. We show that transfected cells exhibit increased proliferation and migration in a wound injury model. We confirmed that our therapy provides spatiotemporal control of target gene expression, which is important to avoid possible undesired effects of protein accumulation. Further in vivo studies are warranted to explore the translational impact of this therapy.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.