Downregulation of KAT II in Retinal Ganglion Cells via Intravitreal or Retrograde Delivery of Antisense Oligonucleotides

Abstract

Abstract: : Purpose: It has been shown, that antisense oligonucleotides (as–ODNs) are effective in downregulating gene expression in the retina after intravitreal injection. However, using this method, it is not possible to regulate the expression of genes only in retinal ganglion cells (RGC). Therefore, the aim of the present study was to investigate changes of kynurenine–aminotransferase II (KAT II) and kynurenic acid (KYNA) content in RGC after intravitreal versus retrograde delivery of as–ODNs. Methods: RCG were transfected with fluorescently labeled oligonucleotides via injections to both superior colliculi in rats. Alternatively, fluorescently labeled oligonucleotides were injected into the vitreous bodies of another group of animals. Immunohistochemistry was performed to show the downregulation of KAT II, a gene expressed in retinal ganglion cells, after injections of unlabeled as–ODNs and scrambled ODNs (controls) in the superior colliculi and intravitreally. Total retinal KYNA content was measured by HPLC. Additionally, the expression of KAT II is currently analyzed by real–time PCR. Results: Following intravitreal injections of fluorescent oligonucleotides, all retinal layers have been accessed, whereas injections in the superior colliculi resulted in a transfection of the retinal ganglion cell layer only. Downregulation of KAT II was recorded using immunohistochemistry after retrograde application as well as after intravitreal injections. Downregulation was equally effective in both tested ways of application. Retinal KYNA content decreased significantly (p=0.01) after antisense oligonucleotide treatment (67.03 ±33 pmol/g wet wt) if compared to controls (103.05±7.05 pmol/g wet wt). Conclusions: These results demonstrate that retrograde transfer of oligonucleotides into retinal ganglion cells is feasible and provides a reliable method to downregulate certain target genes. This technique may become a useful tool to modulate gene expression in the retinal ganglion cell layer in vivo without direct transfer of as–ODNs to other retinal cell layers. Further studies will show the effect of KAT II downregulation on retinal function and morphology.

Keywords: ganglion cells • retina: proximal (bipolar, amacrine, and ganglion cells) • neuro-ophthalmology: optic nerve