(K-432) Pre-formed vesicle-mediated delivery of messenger RNA for retinal gene therapy

Preparation of initial pre-formed lipid vesicles (PFVs) involved standard microfluidic mixing of aqueous and lipid solutions at a 3:1 volumetric ratio. After mixing, PFVs were dialyzed against a citrate buffer (50 mM, pH 4) for 24 hours and subsequently concentrated. PFVs then underwent a second microfluidic mixing step, combining them with respective mRNA solutions (Fluc, Cre, EGFP) at a 3:1 volumetric ratio to yield PFV-LNPs. The PFV-LNPs were dialyzed twice using phosphate buffer saline (PBS, pH 7.4). For comparison, standard LNPs were prepared using a two-phase microfluidic mixing method as a control. Characterization of the LNPs included assessing hydrodynamic size, charge, cargo encapsulation efficiency, lipid packing, and CryoTEM to visualize morphology.

To evaluate transfection and endosomal escape capabilities, Fluc mRNA-containing PFV-LNPs were tested in four cell lines: HeLa (human cervical epithelial cells), ARPE19 and 661W (retina cell models), and HEK293T/17 cells. Additionally, mRNA transfection was examined in human induced pluripotent stem cell (iPSC) retinoids.

After confirming successful transfection in various models, intravenous and subretinal mRNA delivery was assessed in BALB/c, Ai9, and 129X1 murine models. Subretinal injections were specifically performed in Ai9 and 129X1 models. Furthermore, subretinal mRNA delivery was then evaluated in non-human primate models.

In this study, we explored the applicability of solvent-free mixing for encapsulating mRNA in lipid-based nanoparticles (LNPs) and delivering them to the posterior segment of the eye. Lipid-based platforms for mRNA delivery require distinct considerations compared to small interfering RNA (siRNA), as mRNA molecules are longer, single-stranded, and possess more accessible phosphate groups.

We observed significant deviations in size, architecture, and lipid density at the lipid membrane-nucleic acid interface of PFV-LNPs, which result from the formation of large pockets containing mRNA and a more hydrated lipid membrane. This unique morphology during the fusion-rupture event of mRNA-lipid complexation differs from other nucleic acid carriers.

Furthermore, our investigation highlights intriguing insights into the nature of mRNA-lipid complexation during the fusion-rupture event with potential to confer unique advantages for delivery. Notably, this distinctive morphology was beneficial for local administration, particularly for subretinal injection of mRNA. These findings underscore the importance of cargo packing in the development of future non-viral retinal therapeutics. Overall, our study provides valuable insights into the behavior of mRNA-lipid complexes and displays the potential of the solvent-free mixing approach for retinal gene therapy.

This project was supported through funding from the National Heart Lung and Blood Institute (NHLBI) 1R01HL146736-01 (G.S.), SAHAY 19XX0 (G.S.), and the National Eye Institute (NEI) R21EY031066-02.