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Samantha R. De Silva, Daniel M. Lipinski, Peter Charbel Issa, Mandeep S. Singh, Nathan J. Walker, Mark W. Hankins, Robert E. MacLaren; Assessment Of Adeno-associated Virus Serotypes In Transducing Human Retina Using An Ex-vivo Tissue Culture System. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1887.
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Gene therapy for the treatment of retinal degenerations using adeno-associated virus (AAV) is a promising therapeutic option. In order to target further conditions, new vectors must be developed and tested. Alongside pre-clinical testing on mouse or primate models, the ability to test on human retinal tissue would greatly advance the assessment of tropism and efficacy of new AAV vectors.
Retinal detachment surgery in some patients involves removing a small area of retina (retinectomy). Ethical approval was obtained to extract and culture this tissue, which would otherwise be disposed of (REC reference no.10/H0505). Research was conducted in compliance with the Declaration of Helsinki.Samples were obtained from 2 patients with their consent and quickly placed into an organotypic culture system which was used to maintain the retinal tissue ex-vivo. AAV serotypes 2/2, 2/5 and 2/8 the latter containing the Y733F capsid mutation were tested. All contained a construct encoding green fluorescent protein (GFP) under a chicken beta actin (CBA) promoter. 10μl of virus (of titre 1x1012 viral particles/ml) was added to each retinal sample and the samples were imaged daily.
We were able to maintain viable human retinal explants for 11 to 14 days using the culture system. Photoreceptor transduction was demonstrated with all vectors tested, with Müller cells also being transduced by AAV 2/2 and the Y733F capsid mutant AAV2/8. The latter vector also successfully transduced bipolar and horizontal cells.
Culturing human retinectomy tissue ex-vivo is an effective model for testing AAV vectors on human tissue. We demonstrate effective human retinal transduction using AAV 2/2 and 2/5, and also with the capsid mutant vector AAV 2/8 Y733F.
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