AAV2-sFLT01 is a vector that expresses a modified soluble Flt1 receptor designed to neutralize the proangiogenic activities of vascular endothelial growth factor (VEGF) for treatment of age-related macular degeneration (AMD) via an intravitreal injection. Owing to minimal data available for the intravitreal route of administration for adeno-associated virus (AAV), we initiated a 12-month safety study of AAV2-sFLT01 administered intravitreally at doses of 2.4 × 10(9) vector genomes (vg) and 2.4 × 10(10) vg to cynomolgus monkeys. Expression of sFlt01 protein peaked at ~1-month postadministration and remained relatively constant for the remainder of the study. Electroretinograms, fluorescein angiograms, and tonometry were assessed every 3 months, with no test article-related findings observed in any group. Indirect ophthalmoscopy and slit lamp exams performed monthly revealed a mild to moderate but self-resolving vitreal inflammation in the high-dose group only, which follow-up studies suggest was directed against the AAV2 capsid. Histological evaluation revealed no structural changes in any part of the eye and occasional inflammatory cells in the trabecular meshwork, vitreous and retina in the high-dose group. Biodistribution analysis in rats and monkeys found only trace amounts of vector outside the injected eye. In summary, these studies found AAV2-sFLT01 to be well-tolerated, localized, and capable of long-term expression.

Inhibition of vascular endothelial growth factor (VEGF) for the management of the pathological ocular neovascularization associated with diseases such as neovascular age-related macular degeneration is a proven paradigm; however, monthly intravitreal injections are required for optimal treatment. We have previously shown that a novel, secreted anti-VEGF molecule sFLT01 delivered by intravitreal injection of an AAV2 vector (AAV2-sFLT01) gives persistent expression and is efficacious in a murine model of retinal neovascularization. In the present study, we investigate transduction and efficacy of an intravitreally administered AAV2-sFLT01 in a nonhuman primate (NHP) model of choroidal neovascularization (CNV). A dose-dependent and persistent expression of sFLT01 was observed by collecting samples of aqueous humor at different time points over 5 months. The location of transduction as elucidated by in situ hybridization was in the transitional epithelial cells of the pars plana and in retinal ganglion cells. AAV2-sFLT01 was able to effectively inhibit laser-induced CNV in a dose-dependent manner as determined by comparing the number of leaking CNV lesions in the treated versus control eyes using fluorescein angiography. Our data suggest that intravitreal delivery of AAV2-sFLT01 may be an effective long-term treatment for diseases caused by ocular neovascularization.

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A novel two-step real-time RT-PCR assay using SYBR Green I was developed for the detection of acute Bovine Viral Diarrhoea virus (BVDV) infection in whole blood from cattle. During infection animals experience a characteristic transient leucopenia and the number of cells per volume of blood changes over time; so quantitation of viral load by reference to a cellular housekeeping gene is not ideal as this may hide significant animal to animal variation. Therefore, to facilitate comparison of different samples, an external RNA reference was used for normalisation whereby each sample was spiked with the RNA virus, Canine Enteric Coronavirus (CECov), prior to RNA extraction, for comparative purposes. Real-time RT-PCR was carried out with two primer sets designed to amplify either a 156 bp region of the BVDV 5'-UTR or a 280 bp region of the CECov nucleocapsid protein gene. Linearity and efficiency of the assay was established and the method assessed using samples from BVDV-challenged calves. Viral RNA was quantified on days 6 and 14 post-challenge by real-time RT-PCR. Infectious virus isolation by traditional cell culture was negative after day 7. This study demonstrates encouraging results for rapid, sensitive and reliable detection of acute BVDV infection and provides an alternative real-time RT-PCR method for use on whole blood samples or samples where suitable housekeeping genes are not available.

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