Abstract
Purpose : Age-related macular degeneration (AMD) is the most common cause of central vision loss globally. Patients with nAMD treated with regular intraocular injections of anti-VEGF therapies, to which some patients are unresponsive, exhibit non-compliance and risk complications. Interleukin-33 (IL-33) is a promising new treatment for AMD. We have investigated complexion, transfection efficiency and ocular penetration of our interleukin-33 (IL-33) mRNA nanoparticle (NP) eye drop to treat nAMD.
Methods : We tested IL-33 mRNA+NP (500ng mRNA) complexion using zeta potentials. Transfection efficiency was tested in vitro with nanoparticles (N:P 6,8,10) in ARPE-19 and human ocular choroidal fibroblasts (HOCFs) using GFP mRNA+NP and measured FITC+ using flow cytometry. We tested cell toxicity and viability using MTT and LDH assays. We tested the penetration of our eye drop in ex vivo porcine assay and in vivo mouse eyes, 1h after applying 500ng of mRNA therapeutic by quantifying distribution in the vitreous and retina/RPE/sclera using RT-PCR.
Results : We have shown successful complexion of IL-33 mRNA+NP through high charge and small diameter, with a mean diameter of 114.6 ± 10.1 nm (± SD), zeta potential charge of 15.7 ± 5 mV for N:P 10. There was successful translation of our GFP mRNA+NP in ARPE-19 and HOCFs, with the greatest FITC+ cells with N:P 10 of our NPs (4.56% ± 0.29% FITC+ cells compared to 0.09% ± 0.01% in cells treated with GFP mRNA alone, P<0.0001). We show no toxicity with our mRNA+NP complexes. Excitingly, we have shown penetration of our IL-33 mRNA+NP into the vitreous (~0.5pg) and retina (~0.2pg) of an ex vivo porcine eye. In the mouse eye in vivo, we detected 2.45±3.34 ng of IL-33 mRNA when applied as a topical IL-33 mRNA+NP treatment compared to 0.23±0.18 ng detected in EGFP mRNA+NP control group (unpaired t-test P=0.881). High levels of IL-33 mRNA was detected in eyes receiving intravitreal injection of IL-33 mRNA+NP (16.71±3.69ng) which was considerably less when IL-33 mRNA was uncomplexed (0.0043±0.001ng), suggesting that our NP protects the mRNA from degradation.
Conclusions : Our IL-33 mRNA+NP immunotherapeutic offers an exciting new topical therapeutic for AMD and offers the potential for delivery to the retina/RPE/choroid. Further experiments will test our IL-33 mRNA+NP in a mouse in vivo L-CNV model.
Methods : We tested IL-33 mRNA+NP (500ng mRNA) complexion using zeta potentials. Transfection efficiency was tested in vitro with nanoparticles (N:P 6,8,10) in ARPE-19 and human ocular choroidal fibroblasts (HOCFs) using GFP mRNA+NP and measured FITC+ using flow cytometry. We tested cell toxicity and viability using MTT and LDH assays. We tested the penetration of our eye drop in ex vivo porcine assay and in vivo mouse eyes, 1h after applying 500ng of mRNA therapeutic by quantifying distribution in the vitreous and retina/RPE/sclera using RT-PCR.
Results : We have shown successful complexion of IL-33 mRNA+NP through high charge and small diameter, with a mean diameter of 114.6 ± 10.1 nm (± SD), zeta potential charge of 15.7 ± 5 mV for N:P 10. There was successful translation of our GFP mRNA+NP in ARPE-19 and HOCFs, with the greatest FITC+ cells with N:P 10 of our NPs (4.56% ± 0.29% FITC+ cells compared to 0.09% ± 0.01% in cells treated with GFP mRNA alone, P<0.0001). We show no toxicity with our mRNA+NP complexes. Excitingly, we have shown penetration of our IL-33 mRNA+NP into the vitreous (~0.5pg) and retina (~0.2pg) of an ex vivo porcine eye. In the mouse eye in vivo, we detected 2.45±3.34 ng of IL-33 mRNA when applied as a topical IL-33 mRNA+NP treatment compared to 0.23±0.18 ng detected in EGFP mRNA+NP control group (unpaired t-test P=0.881). High levels of IL-33 mRNA was detected in eyes receiving intravitreal injection of IL-33 mRNA+NP (16.71±3.69ng) which was considerably less when IL-33 mRNA was uncomplexed (0.0043±0.001ng), suggesting that our NP protects the mRNA from degradation.
Conclusions : Our IL-33 mRNA+NP immunotherapeutic offers an exciting new topical therapeutic for AMD and offers the potential for delivery to the retina/RPE/choroid. Further experiments will test our IL-33 mRNA+NP in a mouse in vivo L-CNV model.
| Original language | English |
|---|---|
| Journal | Investigative Opthalmology and Visual Science |
| Volume | 64 |
| Issue number | 8 |
| Publication status | Published - 01 Jun 2023 |
| Event | Association for Research in Vision and Ophthalmology Annual Meeting 2023 - Ernest N. Morial Convention Center, New Orleans, United States Duration: 23 Apr 2023 → 27 Apr 2023 |