In vivo delivery of phosphorothioate oligonucleotides into murine retina

Objectives: To determine the fate of phosphorothioate oligonucleotides (S-ODNs),which are commonly used for antisense strategy, in murine retina in vivo with the use of fluorescein isothiocyanate (FITC)-labeled S-ODNs, and to evaluate our fusogenic liposome system that may facilitate the delivery of S-ODNs. Methods: The FITC-labeled S-ODNs were encapsulated in liposomes, which were then coated with the envelope of inactivated hemagglutinating virus of Japan (HVJ; Sendai virus) by fusion (HVJ liposomes). Intravitreal injection of naked FITC-labeled S-ODNs or of the HVJ liposomes was done in ICR mice. After fixation, cryosections and flat-mounted retinas were prepared and examined by fluorescence microscopy. Results: Injection of naked FITC-labeled S-ODNs at 3 mu mol/L exhibited weak fluorescence in 13% of the cells in the ganglion cell lay er, When the concentration was increased to 30 mu mol/L, high fluorescence was seen in 59% of cells in the ganglion cell layer at this time, This fluorescence diminished within a day. In contrast, injection of HVJ liposomes containing FITC-labeled S-ODNs at 3 mu mol/L resulted in high fluorescence in 44% of the cells in the ganglion cell layer at 1 hour, and this fluorescence lasted for up to 3 days, This treatment also resulted in high fluorescence within retinal vessel walls, and weak fluorescence in photoreceptor cells. Conclusions: Intravitreally injected S-ODNs were rapidly eliminated from neural retina, and the use of HVJ liposomes could improve the delivery of S-ODNs. This method may be a potentially useful system for the antisense-based therapies for retinal diseases.