Characterization of Regional RTP801 Gene Expression Within the Retina and the Concentration-Effect Relationship of PF-655, an RTP801-silencing siRNA, Following Intravitreous Administration to Diabetic Rats

Abstract

Purpose: : RTP801 is a hypoxia-induced stress response gene implicated in retinopathy. PF-655 is a 19-mer siRNA targeting a sequence in the RTP801 gene, and is currently in Phase II clinical trials for the treatment of wet AMD and DME. Previous studies demonstrated that PF-655 silenced RTP801 gene expression in retina and retinal pigmented epithelium/ choroid (RPE/C) of rat models of diabetic retinopathy and CNV¹. This study was conducted: (1) To localize regionally enriched intraocular tissue expression of RTP801 using laser capture microdissection (LCM) followed by qPCR analysis, and (2) to characterize the PK/PD relationship of PF-655-mediated RTP801 gene down-regulation.

Methods: : (1) LCM was performed on naïve rat retina (ganglion cell layer and the outer plexiform layer (OPL) with approximately one-third of adjacent inner nuclear layer (INL) and outer nuclear layer (ONL)) and choroid. These sections were then analyzed for RTP801 expression using qPCR. (2) Satellite rats (n=21) were administered a single, unilateral intravitreous dose of PF-655 (50 µg/eye). Ocular tissues from these rats were collected over a 10 day interval postdose, and PF-655 concentrations determined. PK/PD modeling (using results from this and previous studies) was employed to characterize the relationship between the ocular PK of PF-655 and the onset and duration of gene-silencing activity.

Results: : RTP801 expression was enriched in LCM samples obtained from INL/OPL/ONL region of the normal retina. Appreciably less expression was observed in LCM sections of the choroid and ganglion cell layers. PF-655-mediated gene-silencing effect in ocular tissues was maintained well after most of the retina and vitreous drug levels were cleared, and was best characterized by an effect compartment PK/PD model to account for the lag time of effect.

Conclusions: : RTP801 retinal expression patterns were higher in regions containing retinal vasculature and were consistent with results from a murine oxygen induced retinopathy model². Prolonged suppression of RTP801 expression was observed in the absence of quantifiable concentrations of PF-655, consistent with RISC mechanism of siRNA gene-silencing._{1. IOVS 2010; S6448 Late Breaking Abstracts; 2. IOVS 2004;45:3796-3805}

Keywords: gene/expression • diabetic retinopathy • retina