Purchase this article with an account.
Katrien Remaut, Joke Devoldere, Lies De Groef, Karen Peynshaert, Heleen Dewitte, Lieve K M Moons, Stefaan De Smedt; Potential and pitfalls of intravitreal injected mRNA as ocular neuroprotection strategy. Invest. Ophthalmol. Vis. Sci. 2018;59(9):6007. doi: https://doi.org/.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Retinal cell degeneration is a leading cause of vision loss. The local expression of neurotrophic factors by the delivery of messenger RNA (mRNA) to the retina could halt retinal cell degeneration. Modifications in the mRNA backbone have enhanced the stability and duration of protein expression. Nevertheless, the efficient delivery of mRNA remains a major bottleneck. Here, we evaluated the potential of mRNA delivery to the retina after intravitreal injection. Therefore, mRNA modifications and nanoparticles were first optimized in vitro to transfect Müller cells in the presence of vitreous. Then, selected formulations were injected ex vivo and in vivo to assess the amount and extent of protein expression.
MIO-M1 cells were seeded in a transwell system and transfected in the presence of vitreous isolated from fresh bovine eyes, using GFP mRNA delivered by the commercial carrier MessengerMAX®. The duration and level of protein expression was followed by flow cytometry for different mRNA concentrations and modifications. The carriers’ mobility in vitreous was determined by single particle tracking (SPT). In vivo, mRNA was injected intravitreal or subretinal and GFP expression was assessed after 2 or 7 days. Also the potential to transfect ex vivo bovine retinal explants was evaluated using luciferase expressing mRNA.
MessengerMAX formed nanoparticles with mRNA that were able to transfect >80% of Müller cells in culture, without major effect of the presence of vitreous. The m1ψU(1.0) mRNA modification outperformed other mRNA modifications, resulting in a 25-fold higher protein expression. SPT analysis revealed that the negatively charged messengerMAX lipoplexes showed a high intravitreal mobility and were able to result in luciferase protein expression when intravitreal injected into our ex vivo bovine retinal explant model with vitreous attached. Using GFP expressing mRNA, however, the GFP expression level compared to background is more difficult to detect, both ex vivo and in vivo.
Overall, we conclude that messengerMAX lipoplexes complex mRNA, are stable in vitreous, and have the ability to deliver mRNA to the retina after intravitreal injection. As the expression levels ex vivo and in vivo are much lower when compared to in vitro Müller cell culture, however, we will investigate further how to overcome the delivery bottlenecks for mRNA after intravitreal injection.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
This PDF is available to Subscribers Only