Li Weiran, Vanluchene Helena, Raes Laura, Peynshaert Karen, Veys Lien, Hernández Sofía González, De Lombaerde Emily, De Geest Bruno G, Sanders Niek N, Moons Lieve, Raemdonck Koen, De Groef Lies, Remaut Katrien
Ghent Research Group on Nanomedicines, Laboratory of General Biochemistry and Physical Pharmacy, Faculty of Pharmaceutical Sciences, Ghent University, Ottergemsesteenweg 460, 9000 Ghent, Belgium.
Cellular Communication and Neurodegeneration Research Group, Animal Physiology and Neurobiology Division, Department of Biology, Leuven Brain Institute, KU Leuven, Naamsestraat 61, Box 2464, 3000 Leuven, Belgium.
J Control Release. 2025 Jul 12;385:114027. doi: 10.1016/j.jconrel.2025.114027.
Messenger RNA (mRNA) therapeutics offer a powerful and versatile approach for treating retinal diseases by enabling transient expression of therapeutic proteins in the retina. By delivering carefully designed mRNAs, it is possible to restore, replace, or modulate gene function, opening new avenues for regenerative therapies and vision preservation. Lipid nanoparticles (LNPs) are the current state-of-the-art delivery system for mRNA. While the use of mRNA LNPs as vaccines has been booming, only few research groups have investigated LNP-mediated delivery of mRNA to the retina in vivo, with as main focus the amount and location of mRNA expression in the retina. Alternative mRNA platforms such as self-amplifying RNA (saRNA) have not been investigated in the eye before. Also the immunogenicity associated with retinal mRNA or saRNA LNP delivery remains largely unexplored. In this work, we explored the efficacy and immunogenicity of intravitreally delivered mRNA and saRNA LNPs to the mouse retina, using the established ionizable lipids C12-200 and DLin-MC3-DMA, and a recently developed ionizable lipid S-Ac7-DOg. We found that in vitro, C12-200 and S-Ac7-DOg LNPs outperformed DLin-MC3-DMA LNPs in terms of mRNA expression in retinal cells. In vivo, mRNA expression remained limited to the optic nerve head region and a few Müller glia in the retina for all LNPs tested, while saRNA expression was almost completely absent. In vitro only the saRNA LNPs triggered innate immunity, in the order C12-200 > MC3 > S-Ac7-DOg, while in vivo the ionizable lipid C12-200 triggered the highest, but transient immune response, followed by MC3 and S-Ac7-DOg. Overall, we conclude that intravitreal delivery of mRNA and saRNA LNPs currently results in a limited protein expression, independent of LNP composition and cargo. In addition, LNP composition plays a crucial role in triggering a local immune response, with C12-200 being the most immunogenic and S-Ac7-DOg the least immunogenic ionizable lipid. These findings highlight that both immunogenicity and delivery efficiency to the retina remain key challenges to address in future optimization efforts.
信使核糖核酸（mRNA）疗法为治疗视网膜疾病提供了一种强大且通用的方法，可使治疗性蛋白质在视网膜中短暂表达。通过递送精心设计的mRNA，有可能恢复、替代或调节基因功能，为再生疗法和视力保护开辟新途径。脂质纳米颗粒（LNP）是目前用于mRNA的最先进递送系统。虽然mRNA-LNP作为疫苗的应用蓬勃发展，但只有少数研究小组在体内研究了LNP介导的mRNA向视网膜的递送，主要关注视网膜中mRNA表达的量和位置。此前尚未在眼部研究过诸如自我扩增RNA（saRNA）等替代mRNA平台。与视网膜mRNA或saRNA-LNP递送相关的免疫原性也在很大程度上未被探索。在这项工作中，我们使用已确立的可电离脂质C12-200和DLin-MC3-DMA以及最近开发的可电离脂质S-Ac7-DOg，探索了玻璃体内递送mRNA和saRNA-LNP至小鼠视网膜的疗效和免疫原性。我们发现，在体外，就视网膜细胞中的mRNA表达而言，C12-200和S-Ac7-DOg-LNP比DLin-MC3-DMA-LNP表现更优。在体内，对于所有测试的LNP，mRNA表达仅限于视神经乳头区域和视网膜中的少数穆勒胶质细胞，而saRNA表达几乎完全缺失。在体外，只有saRNA-LNP触发了先天免疫，顺序为C12-200 > MC3 > S-Ac7-DOg，而在体内，可电离脂质C12-200引发了最高但短暂的免疫反应，其次是MC3和S-Ac7-DOg。总体而言，我们得出结论，目前玻璃体内递送mRNA和saRNA-LNP导致的蛋白质表达有限，与LNP组成和所载物无关。此外，LNP组成在触发局部免疫反应中起关键作用，C12-200是免疫原性最强的可电离脂质，而S-Ac7-DOg是免疫原性最弱的可电离脂质。这些发现凸显出免疫原性和向视网膜的递送效率仍是未来优化工作中需要解决的关键挑战。
