Modulation of Gene Expression in the Eye with Antisense Oligonucleotides

Abstract

One advantage of antisense oligonucleotides (ASOs) for drug development is their long-lasting gene knockdown after administration in vivo. In this study, we examine the effect on gene expression after intraocular injection in target tissues in the eye. We examined expression levels of the Malat1 gene after intracameral or intravitreal (IV) injection of an anti-Malat1 ASO in corneal epithelium/stroma, corneal endothelium, lens capsule epithelium, neurosensory retina, and retinal pigment epithelium/choroid of the mouse eye. We assessed potency of the compound at 7 days as well as duration of the gene knockdown at 14, 28, 60, 90, and 120 days. The ASO was more potent when delivered by IV injection relative to intracameral injection, regardless of whether the tissues analyzed were at the front or back of the eye. For corneal endothelium, inhibition was >50% after 120 days for ASO at 50 μg. At IV dosages of 6 μg, we observed >75% inhibition of gene expression in the retina and lens epithelium for up to 120 days. ASOs have potential as long-lasting gene knockdown agents in the mouse eye, but efficacy varies depending on the specific ocular target tissue and injection protocol.

Keywords: antisense oligonucleotide; cornea; intraocular; retina.

FIG. 1.

Antisense oligonucleotides (ASOs) and experimental scheme. (A) MOE-gapmer ASOs used in this study. (B) Eye anatomy and intraocular injection methods used in mouse eye. (C) Experimental design for this study, showing dissected tissues used to obtain RNA for qPCR. C:5-methylcytidine.

FIG. 2.

Malat1 RNA expression in ocular tissues after intracameral or IV injection of ASO. Malat1 gene expression in (A) Corneal epithelium/stroma. (B) Corneal endothelium. (C) Lens capsule epithelium. (D) Neurosensory retina. (E) RPE/choroid 7 days after intracameral (IC) injection of different doses of anti-Malat1 gapmer ASO compared with IV injection. All injections were performed using 1 μL total volume. Error bars represent SEM. *P < 0.05; **P < 0.01; ***P < 0.001 relative to noncomplementary control (MC) by Student's t-test. IV, intravitreal; RPE, retinal pigment epithelium; SEM, standard error of the mean.

FIG. 3.

Dose–response of anti-Malat1 gapmer ASO (gMal) in ocular tissues delivered by IV injection at 0.5, 1, 3, 6, 12, 25, or 50 μg. qPCR results of Malat1 expression for (A) Corneal epithelium/stroma. (B) Corneal endothelium. (C) Lens capsule. (D) Retina. (E) RPE/Choroid. Eyeballs were harvested 7 days after IV injection of different dosages of ASO, and then dissected for analysis. Error bars represent SEM. *P < 0.05; **P < 0.01; ***P < 0.001 relative to noncomplementary control (MC) by Student's t-test.

FIG. 4.

Duration study of anti-Malat1 gapmer ASO (gMal) in eye tissues, 14, 18, 60, 90, and 120 days. Malat1 expression was analyzed in (A) Corneal epithelium/stroma. (B) Corneal endothelium. (C) Lens capsule. (D) Retina. (E) RPE/Choroid after indicated time after a 6 or 50 μg injection. Error bars represent SEM. *P < 0.05; **P < 0.01; ***P < 0.001 relative to noncomplementary control (MC) by Student's t-test.