Dynamics of phosphorothioate oligonucleotides in normal and laser photocoagulated retina

Abstract

Aims: To investigate the distribution, persistence, and stability of fluorescently labelled phosphorothioate oligonucleotides (PS-ODNs) in normal and laser photocoagulated retina following intravitreal injection in the rat.

Methods: Fluorescently labelled PS-ODNs were injected intravitreally into pigmented eyes at doses of 0.5-10.0 nmol in 2.0 microl solution. The dynamics of PS-ODNs was evaluated by fluorescent microscopy of cryosections and flat mounted retinal pigment epithelium (RPE)-choroid-sclera. Genescan analysis was used to assess the integrity of PS-ODNs in the retina after injection. The dynamics of PS-ODNs was also evaluated in the retina following krypton laser photocoagulation with a protocol producing choroidal neovascularisation (CNV).

Results: Following intravitreal injection the PS-ODNs demonstrated dose and time dependent distribution and persistence in the retina, where they accessed all neural layers. However, they preferentially accumulated in the RPE layer, demonstrated as bright granules in the cytoplasm of the cells. Injections of 5.0 and 7.5 nmol of PS-ODNs exhibited strong fluorescence in the retina for 6 weeks after injection. Genescan analysis demonstrated that the PS-ODNs remained almost completely intact for at least 12 weeks. Following laser treatment, the PS-ODNs were concentrated in the regions of laser photocoagulation and retained high intensity for at least 8 weeks after injection, particularly localised to macrophages, RPE, and the local choroidal tissue.

Conclusions: These results indicate that PS-ODNs are stable and accessible to most neural layers of the retina, and they preferentially accumulate in the RPE layer following intravitreal injection. The successful delivery of PS-ODNs into normal and laser photocoagulated retina suggests that PS-ODNs may have potential in the development of therapy for attenuating retinal degenerations and CNV.

Figure 1

Distribution and persistence of PS-ODNs (DS 012) in the retina 2 weeks after intravitreal injection (A-D). (A) 10.0 nmol, (B) retinal morphology of (A), counterstaining with haemotoxylin. The arrows indicate the RPE layer. (C) 2.0 nmol, the arrows are pointing to the nuclei of RPE cells. Inset: cytoplasmic distribution of PS-ODN in RPE observed by confocal laser scanning microscopy. (D) 0.5 nmol. (E and F) 5.0 nmol, 6 weeks (E) and 8 weeks (F) after injection. (G and H) Fluorescent microscopic images from the control eyes injected with fluorescent 6-FAM at 1 hour (G) and 24 (H) hours after injection. The fluorescent 6-FAM was mostly confined to the vitreous but some just entered into the ganglion cell layer at 1 hour after injection (G). However, no fluorescent signal was detected in the neural retina at 24 hours after injection (H). Original magnifications: (A-H) ×50; inset of (C) ×100. RPE = retinal pigment epithelium; POS = photoreceptor outer segment; ONL = outer nuclear layer; INL = inner nuclear layer; GCL = ganglion cell layer; ILM = inner limiting membrane.

Figure 2

Distribution of fluorescently labelled PS-ODN (DS 012, 5.0 nmol) in the RPE cells by fluorescent microscopy of flat mounted RPE-choroid-sclera. (A) Two weeks after injection, the fluorescently labelled PS-ODNs appear as bright granules in the cytoplasm of the RPE cells. (B) Eight weeks after injection. (C) Microphotograph of the hexagonal RPE cells in (A), observed by light microscopy. (D) A control eye injected with 5.0 nmol of sodium fluorescein, 2 week after injection. Original magnification (A-D) ×100.

Figure 3

Accurate Genescan sizing of injected and uninjected PS-ODN using oligonucleotide size standards. (A) The original 20-mer DS 012 was compared directly with a series of PS-ODNs varying in size from 15 to 25 nucleotides in length and their electrophoretograms superimposed using the internal standard as a reference. Oligonucleotides within this range of size were clearly resolved using the Genescan analysis software under the altered run conditions. (B) DNA extracted from the retina of an eye 12 weeks following injection with DS 012 for 12 weeks was run and compared directly with the oligonucleotide size standard. The extracted oligonucleotide demonstrated some minor peaks representing N-1 degradation products.

Figure 4

Persistence of PS-ODN following intravitreal injection demonstrated by Genescan analysis. Electrophoretograms of DNA extracted from the retina (A, C, and E) and the RPE-choroid-sclera complex (B, D, and F) at 6 weeks (A, B), 8 weeks (C, D), and 12 weeks (E, F) after injection with DS 012, and the oligonucleotide size standard (G). The internal standard is superimposed in all electrophoretograms at the same data collection point and the peaks for the PS-ODN are equivalent for both tissue samples at all time points. Note the actual fluorescent intensity of 6 week samples is effectively 130 times higher than that of 8 and 12 week samples.

Figure 5

Distribution of PS-ODNs (DS 012, 5.0 nmol) in the retina following krypton laser photocoagulation. (A, B) Retinal whole mounts, 4 weeks (A) and 8 weeks (B) after injection; the PS-ODNs are concentrated in the regions of laser photocoagulation with an appearance of clumps of bright granules at the sites of laser burns (arrows). (C, D) Cryosections from eyes injected with DS 012 following krypton laser photocoagulation, 4 weeks (C) and 8 weeks (D) after injection. The fluorescent signal is particularly localised to infiltrating cells (arrows) and the RPE layer. (E) Higher magnification of (C) observed by light microscopy, counterstaining with haemotoxylin, the arrows indicating pigment laden infiltrating cells. (F) A serial section of (C) and (E), immunostaining for CD68 (arrows). Original magnification: (A, B) ×10; (C, D) ×50; (E, F) ×100. GCL = ganglion cell layer; INL = inner nuclear layer; ONL = outer nuclear layer; RPE = retinal pigment epithelium.