(K-439) Bio-inspired non-viral gene therapy in the mouse retina

Liposomes were prepared via thin film hydration and extrusion method. Typically, lipid mixtures were dissolved in chloroform and dried under the vacuum. The thin film was then hydrated and mixed with Cre-mRNA. The dispersion of hydrated lipid film and mRNA were extruded through 100 nm Polycarbonate Membrane with mini extruder for twenty-five extrusion cycles. For hyaluronic acid (HA) coating, the liposomes were stirred with HA overnight. The liposomes were characterized by dynamic light scattering (DLS) for liposome physical characterization. The liposomes were injected into Ai14 mouse retina via intravitreal route and the mouse were transcardially perfused with PBS wash and 4% paraformaldehyde. For retinal whole mounting, the retina tissues were isolated and flattened on glass slide.

In this study, we fabricated liposomes with surface modification using hyaluronic acid (HA), which exhibit promising biocompatibility and potential cell/tissue targeting effect. Liposomes were prepared by thin film hydration and extrusion methods. The liposomes size was determined by dynamic light scattering (DLS). The average particle size was about 131.78 ± 10.75 nm, with a PDI (Polydispersity Index) of 0.25 ± 0.01. The successful HA surface modification was demonstrated via surface Zeta potential measurement, which exhibited a slight negative charge at -3.36 ± 0.21 mV (Figure 1B). Then, we evaluated in vivo mRNA expression by intravitreal injection in Ai14 transgenic mice. This mouse model provides a strong red fluorescent signal (Tdtomato) following the Cre-LoxP system to investigate liposomes in vivo efficacy. Liposomes were injected in Ai14 mice via intravitreal injection, following a whole mounting on day 7 post-injection to observe Tdtomato expression. As shown in Figure 1C, Tdtomato expression in the retina was visualized using confocal microscopy on whole-mount retinal preparations. The confocal images revealed distinct Tdtomato fluorescence patterns throughout various retinal layers.
In conclusion, we successfully fabricated HA-coated liposomes for targeted retinal gene therapy. The results demonstrated robust Tdtomato expression induced by Cre mRNA encapsulated liposomes in Ai14 mice. Our study highlights the promising potential of HA-coated liposomes as non-viral gene delivery tools with efficient gene expression for ocular diseases. Further investigations are needed to explore their specific targeting capability within retinal layers.