Abstract
Purpose :
Retinal gene therapy using messenger RNA (mRNA) therapeutics is a promising strategy to induce transient (days-months) protein expression in the retina. However, only few mRNA-lipid nanoparticles reach the retina after intravitreal delivery due to the presence of the vitreous and the inner limiting membrane. We explore the use of self-amplifying mRNA (saRNA) that, as a result of its self-replicative nature can be effective at lower doses, making it an interesting candidate to achieve protein expression in difficult to reach tissues, like the retina.
Methods :
The ability of mRNA and saRNA to induce protein expression was compared using the carrier MessengerMAX® in a dose titration experiment. Furthermore, two strategies were tested to limit immune activation and enhance protein expression: i) cellulose-based purification to remove double-stranded RNA byproducts, ii) type I IFN decoy receptor B18R. All transfections were performed in ARPE-19 and Müller (MIO-M1) cells (eGFP expression, flow cytometry), and ex vivo on bovine explants (luciferase expression, bioluminescent imaging) (n=3). Immune responses were measured with a LEGENDplex assay.
Results :
In the dose titration experiment, we found that a low dose, saRNA outperformed mRNA in terms of eGFP expression, while at a high dose, saRNA seemed to lose its self-replicative advantage as a comparable dose of non-replicative mRNA induced more expression. This was attributed to the immunogenicity of saRNA as a gradual increase in the secretion of innate immune-related cytokines (IL-1β, IL-6, TNF-α, IFN-β, IFN-λ1, IFN-λ2/3) was observed with an increasing dose of saRNA. The immunogenicity of saRNA could be reduced by cellulose purification and addition of B18R. Moreover, cellulose purification of saRNA led to 274x and 66x more eGFP expression in ARPE-19 and MIO-M1 cells respectively, compared to its standardly purified counterpart (silica-membrane columns). B18R increased expression 2.4-times in ARPE-19 cells. Similarly, cellulose purification of saRNA led to a 6-fold increase in retinal luciferase expression in bovine explants.
Conclusions :
Overall, saRNA has potential to induce protein expression in the retina provided that measures to temper its innate immune-stimulating activity, like cellulose-based purification and combination with an immune inhibitor (e.g. B18R), are taken into account.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.