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rAAV.sFlt-1 Gene Therapy Achieves Lasting Reversal of Retinal Neovascularization in the Absence of a Strong Immune Response to the Viral Vector

¹From the Centre for Ophthalmology and Visual Science, and the ⁵School of Animal Biology, and ⁷Western Australian Institute for Medical Research, The University of Western Australia, Crawley, Western Australia, Australia; the ²Department of Molecular Ophthalmology and the ³Centre for Experimental Immunology, Lions Eye Institute, Nedlands, Western Australia, Australia; the ⁴University of Queensland, Centre for Clinical Research and the Perinatal Research Centre, Royal Brisbane and Women’s Hospital, Herston, Queensland, Australia; and the ⁶School of Pharmacy, Curtin University of Technology, Bentley, Western Australia, Australia.

PURPOSE. To determine the efficacy of rAAV.sFlt-1–mediated gene therapy in a transgenic mouse model of retinal neovascularization (trVEGF029) and to assess whether rAAV.sFlt-1 administration generated any deleterious, long-lasting immune response that could affect efficacy.

METHODS. trVEGF029 mice were injected subretinally with rAAV.sFlt-1 or phosphate-buffered saline. Fluorescein angiography and electroretinography were used to compare the extent of fluorescein leakage from retinal vessels and retinal function, respectively. A group of eyes was enucleated, and the retinal vasculature and morphology were studied by confocal and light microscopy. Cells were isolated from the posterior eyecups and spleens of a further group, and immune cell subset populations were investigated by flow cytometry. sFlt-1 protein levels in the eyes were evaluated by ELISA.

RESULTS. After a single rAAV.sFlt-1 injection, sFlt-1 protein levels were upregulated, and there was a reduction in fluorescein leakage from the retinal vessels and an improvement in retinal function. Confocal microscopy of isolectin-IB4-labeled retinal wholemounts showed more normal-appearing capillary beds in rAAV.sFlt-1–injected than in PBS-injected trVEGF029 mouse eyes. Light microscopy demonstrated retinal morphology preservation, with fewer aberrant vessels invading the outer nuclear layer of rAAV.sFlt-1–injected eyes. Furthermore, the immune response to subretinal injection of rAAV.sFlt-1 was limited to a transient increase in CD45⁺ leukocytes that disappeared by 4 weeks after injection. This transient increase was localized to the eye and did not affect long-term therapeutic efficacy.

CONCLUSIONS. The data support the notion that rAAV.sFlt-1 gene therapy is safe and effective for the long-term inhibition of deleterious blood vessel growth in the eye.