Dynamics of phosphorothioate oligonucleotides in normal and laser photocoagulated retina.
OBJECTIVE
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.
Shen
W Y
WY
Department of Molecular Ophthalmology Biology, Lions Eye Institute, Western Australia.
Garrett
K L
KL
da Cruz
L
L
Constable
I J
IJ
Rakoczy
P E
PE
D000818:Animals; D015703:Antigens, CD / Q000378:metabolism; D015214:Antigens, Differentiation, Myelomonocytic / Q000378:metabolism; D016228:Endothelial Growth Factors / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D007267:Injections; D017075:Laser Coagulation; D008222:Lymphokines / Q000378:metabolism; D018613:Microscopy, Confocal / Q000379:methods; D009841:Oligonucleotides / Q000493:pharmacokinetics*; D010857:Pigment Epithelium of Eye / Q000378:metabolism; D051381:Rats; D012160:Retina / Q000378:metabolism*; D013873:Thionucleotides / Q000493:pharmacokinetics*; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D000097382:CD68 Molecule
D015703:Antigens, CD; D015214:Antigens, Differentiation, Myelomonocytic; D016228:Endothelial Growth Factors; D008222:Lymphokines; D009841:Oligonucleotides; D013873:Thionucleotides; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; C067980:CD68 antigen, human; D000097382:CD68 Molecule