Identification and Characterization of Mouse Antisense Oligonucleotides (ASOs) Directed at Rhodopsin

Abstract

Purpose: Antisense technology is a drug discovery platform that uses the availability of genetic information and sequences to treat diseases where small molecules and antibodies may have limited success. The use of antisense technology for the treatment of ocular diseases is an ideal drug development approach that combines specificity, simple formulations (saline) and a long duration of action. The following experiments highlight the discovery and characterization of antisense oligonucleotides (ASOs) targeting the mouse rhodopsin gene.

Methods: In-vitro ASO identification: ASOs were screened for reduction of rhodopsin expression in M-3 cells after electroporation. Rhodopsin mRNA levels were measured by RT-PCR and normalized to GAPDH. In-vivo: Age matched Balb/C or C57BL/6 mice were IVT (intravitreal) injected with 1 ul of various concentrations of ASOs or PBS and sacrificed at various time-points. Eyes were enucleated for histology and retinas wrinkled for RT-PCR RNA analysis.

Results: In-vitro screening identified a number of potent ASOs targeting rhodopsin. Sixteen of the most active ASOs were tested in-vivo by IVT delivery to demonstrate RNA reduction in C57BL/6 mice. Oligo Staining of treated mouse retinas revealed ASO distribution in all the cellular layers of the eyes, including the photoreceptor layer, inner and outer nuclear layers and the RPE. The in vivo screen identified several active ASOs, with ISIS 563334 producing the best rhodopsin mRNA reduction, approximately 60 ± 7% as compared to age matched saline injected eyes 7 days after the 50ug injection. Rhodopsin protein levels measured by IHC showed reductions within the photoreceptor layer and in the outer nuclear layer. Also western blot analysis showed similar reductions to those measured by RNA. Duration of action studies were performed using two ASOs targeting different regions of the mouse rhodopsin gene, demonstrated a long half-life of approximately 2 months following a single IVT injection in C57BLK6 mice.

Conclusions: Antisense technology represents an ideal ocular drug discovery platform. Here we describe the identification of murine ASO targeting rhodopsin gene, the cellular uptake in the rod photoreceptor cells and the long duration of action after a single injection. Our results support the use of ASOs for the treatment retinal disorders.

Keywords: 702 retinitis • 533 gene/expression • 625 opsins