Progressive and irreversible vision loss affects up to 280 million people worldwide, of which 43 million eventually turn blind. Intracellular protein expression by messenger RNA (mRNA) delivery has great potential to treat ocular diseases by cell reprogramming or genome editing. The therapeutic potential of non-viral mRNA delivery by lipid nanoparticles (LNPs) was highlighted in the worldwide vaccination efforts against the Covid19 pandemic. In large contrast, mRNA delivery to the eye is still in its infancy. Theoretically, the eye is however an ‘easy target’, as it is a small, closed compartment that enables local administration. In practice, innate immune responses and permeability barriers at cellular and tissue level create critical bottlenecks. As one of the few research groups worldwide working on ocular mRNA delivery, we identified three major bottlenecks to mRNA delivery upon intravitreal injection. First, nanoparticles should be mobile in the vitreous to migrate to the retina. Second, the inner limiting membrane (ILM) severely blocks LNP transport from the vitreous to the retina. Third, depending on LNP composition, ocular inflammation occurs accompanied with immune recognition by resident macrophages in the vitreous and retina. In this project, we will further screen LNP formulations to obtain 1) sufficient vitreal mobility, 2) a small size to enhance ILM crossing and 3) low inflammatory properties to prevent immune stimulation on the extracellular and intracellular level. Furthermore, we will explore a novel and non-toxic method developed in our lab to digest the ILM with collagenase, resulting in up to 150 fold improvement in mRNA LNP efficacy in preliminary experiments using ex vivo retinal explants. In conclusions, this project will lay the foundations for successful therapeutic mRNA delivery to the retina in vivo, by understanding and defeating intracellular and extracellular delivery barriers and by rationalizing LNP design