Stabilized small interfering RNAs (siRNAs) targeting the vascular endothelial growth factor (VEGF) pathway inhibit angiogenesis in vivo in a rat corneal model

Abstract

Abstract: : Purpose: Small interfering RNAs are short duplex oligonucleotides that can be designed to target and interfere specifically with the expression of disease–related genes. The studies reported here identify and compare the in vivo antiangiogenic effects of stabilized siRNAs that target VEGF or VEGF receptor (VEGFR) mRNAs using a rat model of corneal angiogenesis. Methods: Either VEGF–impregnated filter disks or silver nitrate cauterization was employed to induce angiogenesis in the rat cornea. siRNAs targeting VEGF, VEGFR–1 and VEGFR–2 or control siRNAs were delivered using subconjunctival injection at the time of filter implantation or silver nitrate burn. siRNAs were chemically synthesized and diluted in saline for use. Blood–filled corneal vessels were digitally imaged on day 5 and the neovascular surface area was quantified using computerized morphometry. Results: As an in vivo screen to select active siRNAs for potential therapeutics, ∼40 siRNAs targeting numerous sites in VEGF, VEGFR–1 and VEGFR–2 mRNAs were compared in a series of studies for the ability to inhibit angiogenesis in the rat cornea. In general, antiangiogenic activity in vivo (range 0–40% inhibition) varied empirically with the mRNA site targeted and with the stabilization chemistry employed. For example, treatment with one minimally–modified VEGFR–1 siRNA over the dose range of 0.05 to 1.0 µg siRNA resulted in a 25–36% inhibition of angiogenesis, respectively, while the matched inverted sequence control at all doses averaged <1% inhibition (p=0.001). Completely stabilized siRNA directed at this same site had lower activity, however, targeting a site only a few nucleotides away restored the activity of a completely modified siRNA. Active siRNAs targeting VEGF, VEGFR–1 and VEGFR–2 were selected for further study. Conclusions: Stabilized siRNAs that target VEGF pathway mRNAs significantly inhibit angiogenesis in a rat corneal model of angiogenesis when compared to matched inverted sequence control siRNAs of the same nucleotide composition and stabilization chemistry. For some siRNAs, the addition of modified nucleotides to stabilize the siRNA against degradation in vivo resulted in similar antiangiogenic activity as minimally–modified siRNAs. These data support the therapeutic use of modified siRNAs to inhibit angiogenesis in chronic ocular diseases such as age–related macular degeneration and diabetic retinopathy.

Keywords: neovascularization • age–related macular degeneration • receptors: pharmacology/physiology