Intravitreal injected lipid nanoparticles for efficient and non-immunogenic mRNA delivery to the retina

Abstract

Purpose : Intracellular protein expression by messenger RNA (mRNA) delivery has great potential to treat ocular diseases by cell reprogramming or genome editing. We screened mRNA lipid nanoparticles (LNPs) for (i) the degree, duration and location of protein expression and (ii) the toxicity (immunogenicity) of the formulations after intravitreal injection in mice.

Methods : m1ψU modified mRNA encoding for green fluorescent protein (GFP) was prepared via in vitro transcription, enzymatically polyadenylated and capped with an ARCA cap. eGFP mRNA was formulated into LNPs using microfluidic mixing. LNPs consisted of ionizable lipids (50%), cholesterol (38.5%), helper lipids (10%) and PEGylated lipids (1.5%). Several formulations (based on different ionizable lipids) were screened in ARPE19 cells for particle uptake and transfection efficiency by flow cytometry. LNPs were intravitreally (IVT) injected in mice to determine 1) eGFP expression by confocal scanning laser ophthalmoscopy (cSLO, Heidelberg HRA) and 2) neuroinflammation (i.e. influx of inflammatory cells into vitreous) by optical coherence tomography (OCT, Bioptigen). Cryosections were immunostained for GFAP (glial fibrillary acidic protein indicative of gliosis), Iba1 (Ionized calcium-binding adaptor molecule 1), CD45 (cluster of differentiation 45) and eGFP.

Results : All tested LNPs gave good uptake and transfection efficiency in ARPE19 cells in vitro.
After IVT injection, immunogenicity of LNPs was apparent from a large increase in infiltrating immune cells in the vitreous cavity (Figure 1A, B), with LNP1 being the most and LNP3 the least immunogenic. Immunostaining against Iba1 (Figure 1C, D), confirmed that LNP1 gave the highest microglia activation. Also staining for CD45 and GFAP confirmed that LNP1 was most immunogenic.
eGFP expression was obtained after IVT injection of LNPs (Figure 2), but mostly limited to the optic nerve head (Figure 2B) and a few Müller glia (Figure 2C), with LNP1 and LNP3 giving better results than LNP2.

Conclusions : LNP composition plays a crucial role in triggering an immune response, by currently unknown mechanisms or structure-activity relationships. The tested LNPs currently result in a limited protein expression, localized at the optic nerve head and a few Müller glia. In future research, strategies to limit inflammation and to increase penetration into the retina will be evaluated.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.

 

Figure 1.

 

Figure 2.