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.
