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Joke Devoldere, Karen Peynshaert, Stefaan De Smedt, Katrien Remaut; Intravitreal delivery of chemically modified mRNA for neuroprotection through Müller cell transfection. Invest. Ophthalmol. Vis. Sci. 2016;57(12):6411. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Recent advances in the field of molecular biology have revolutionized mRNA as a therapeutic. As chemical modifications to in vitro transcribed mRNAs reduce its immunogenicity, prolonged protein expression can be obtained, thus broadening the applicability of mRNA in protein replacement therapies. This work aims to evaluate the therapeutic potential of chemically modified mRNA as a therapy for retinal neurodegeneration. In an experimental model based on fresh bovine vitreous, we examined non-viral intra-vitreal delivery of modified reporter mRNA to Müller cells via two lipid-based commercial carriers: messengerMAX and TransIT.
Human Müller glia cells (MIO-M1) were seeded in a transwell system 5 days before transfection. Fresh bovine eyes were obtained from a local abattoir. The vitreous was carefully removed and applied on top of the insert. Lipid-based commercial carriers with modified eGFP mRNA were either delivered to Müller cells via serum-containing medium, injected into the intact vitreous or mixed with enzymatically treated vitreous. Transfection efficiency was quantified by flow cytometry. Cell viability was evaluated both with flow cytometry and a standard MTT assay.
For messengerMAX the percentage of GFP positive cells reached up to 60% upon 24h incubation in serum-containing medium. Transfection levels decreased when transfections were performed in intact or enzymatically treated vitreous, indicating that the vitreous is an important barrier for non-viral mRNA delivery to the retina. Interestingly, although messengerMAX did not show complete mRNA complexation with gel electrophoresis, it proved to be superior over TransIT in all transfection media. However, toxicity results indicated that the TransIT carrier possesses the most favorable safety profile. Therefore, despite its lower transfection potential, the low toxicity could make this carrier suitable in situations where repeated transfections are required.
Our data demonstrate that non-viral lipid based carriers show good potential to transfect Müller cells in vitro. Moreover, chemical modification of the mRNA substantially increased GFP expression in >80% of cultured Müller cells. However, to make these mRNA therapeutics suitable for ocular targets, the development of strategies to overcome the vitreal barrier will be crucial to implement ocular mRNA therapy in the future.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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