Preclinical Evaluation of a Phosphorothioate Oligonucleotide in the Retina of Rhesus Monkey

Abstract

SUMMARY:Overexpression of vascular endothelial growth factor (VEGF) has been strongly implicated in the development of choroidal neovascularization (CNV) in patients with age-related macular degeneration. In this study, a phosphorothioate oligonucleotide (PS-oligo) targeting both human and rat VEGF 165 genes upstream of the translation initiation code, named DS135 in this study, was evaluated for its uptake dynamics and retinal tolerance after intravitreal (IV) and subretinal (SR) injections in the rhesus monkey. Intravitreal and SR injections of a fluorescent-labeled DS135 (FL-DS135) resulted in both dose-and time-dependent uptake and persistence, and FL-DS135 remained detectable in the retina for at least 3 weeks after injection. Ophthalmic examination showed transient vitreous haze after IV delivery of a high dose but not with a low dose of FL-DS135. Histologic examination showed no evidence of retinal degeneration with respect to IV delivery. After SR delivery, however, dose-related cellular infiltration, transient residual fluid, and slight distortion of the neuroretina were observed. The biologic efficacy of DS135 was further assessed in a laser-induced CNV model, and development of CNV was determined by fluorescein angiography and histologic examination. Incomplete inhibition of CNV formation was observed after IV and SR injection of DS135, but no statistically significant difference was achieved when compared with dose-matched control of PS-oligo. Analysis of fluorescein angiogram and histologic examination showed less than 30% incidence of CNV development in this monkey model. Our study demonstrated that PS-oligos can be successfully introduced into the retina, although with potential limitations, after SR delivery. DS135, a PS-oligo targeting the VEGF gene upstream of the translation initiation code, partially inhibited CNV formation. An improved CNV model is necessary for further confirmation of the full therapeutic potency of DS135 before clinical application. (Lab Invest 2002, 82:167-182).

Potential long-term inhibition of ocular neovascularisation by recombinant adeno-associated virus-mediated secretion gene therapy

“…[6][7][8][9][20][21][22] Several studies have further shown that inhibition of VEGF activity by administration of VEGF-specific kinase inhibitors, VEGF antibodies, VEGF receptors and VEGF antisense oligonucleotides successfully suppressed the development of retinal and choroidal NV. [37][38][39][40] In particular, Honda et al 41 showed that expression of adenovirus-mediated sFlt-1 suppressed choroidal NV. However, the adenoviral vector was injected into the rat femoral muscle resulting in systemic circulation of sFlt-1, which dropped to very low levels after 21 days.…”

“…Previous attempts using VEGF antisense oligonucleotides, soluble Flt-1-chimeric proteins and adenovirus-mediated sFlt-1 also failed to completely block retinal or choroidal NV, achieving only a maximum inhibition of 50%. 37,38,40,41 In contrast, administration of kinase inhibitors that blocked phosphorylation by VEGF and platelet-derived growth factor (PDGF) receptors completely prevented the development of retinal NV, but kinase inhibitors of PDGF receptors alone had no effect. 39 Although this implies that blockade of VEGF activity alone is sufficient for the complete inhibition of NV, a synergy might exist between VEGF and PDGF, or other angiogenic factors, which all need to be blocked concurrently for complete inhibition.…”

Preclinical safety evaluation of subretinal AAV2.sFlt-1 in non-human primates

“…The injection procedure was performed under aseptic conditions in a surgical theater equipped for non-human primates. The left eyes of eight non-human primates were subretinally injected with AAV2.sFlt-1 (n ¼ 5), recombinant sFlt-1 protein (n ¼ 1, Cell Sciences, Boston, MA, USA) or AAV2.GFP (n ¼ 2), as previously described, 11,61 but with modifications to the microdelivery system. A 41-gauge subretinal cannula (Bausch and Lomb, Rochester, NY, USA), shortened and fashioned into a bevel tip to allow easier access into the subretinal space, was connected to a flexible pediatric tubing that was in turn connected to a 5-ml syringe.…”

“…The 100 ml virus-or proteincontaining solution was injected into the eye to create a subretinal bleb with a size of about four times the optic disc diameter, as described previously. 11,61 One uninjected non-human primate was used as control.…”

Dendrimers as tunable vectors of drug delivery systems and biomedical and ocular applications

“…In addition, this ODN-1 injected intravitreally in an animal model of CNV led to the attenuation of CNV, as assessed by fluorescein angiography. 73 CNV was induced by laser photocoagulation. 74 The synthesis and use of lipophilic amino acid dendrimers (lipid-lysine) to deliver ODN-1 was accomplished.…”