Human Retinal Explant Culture for Ex Vivo Validation of AAV Gene Therapy

Abstract

Recombinant adeno-associated viral (AAV) vectors have been successfully employed as the mode of gene delivery in several clinical trials for the treatment of inherited retinal diseases to date. The design of such vectors is critical in determining cellular tropism and level of subsequent gene expression that may be achieved following viral delivery. Here we describe a system for living retinal tissue extraction, ex vivo culture, viral transduction and assessment of transgene expression that may be used to assess viral constructs for gene therapy in the human retina at a preclinical stage.

Retinal explant culture: A platform to investigate human neuro‐retina

“…Even within human retina, considerable difference in the AAV‐mediated viral transduction was observed between foveal cones and peripheral cones in a study aiming to transduce foveal cone photoreceptors . Despite these differences, AAV‐mediated viral transduction was achieved in retinal tissues obtained from live donors post‐surgery …”

“…A number of supplements, including hormones, growth factors, serum substitutes, cofactors, lipids and buffers have been included in the retinal explant culture media to promote the growth of explants, especially retinal neurons, whilst maintaining the pH. Glutamine, B‐27 and N2 are the most commonly used supplements in human retinal explant culture . A number of growth factors like neuronal growth factor, fibroblast growth factor and epidermal growth factor have been incorporated in the retinal culture media, specifically to enhance the survival of retinal neurons.…”

“…Also, the differences in cellular distribution across the retina makes it challenging to dissect experimentally reproducible retinal fragments, especially for surgically naïve laboratory personnel. Therefore, internal controls are one of the most important components of a post‐mortem experimental study, but these will be difficult if not impossible to obtain in cases of live donors where the retinal tissue is usually diseased, of low volume or oxygen deprived …”

“…105 Despite these differences, AAV-mediated viral transduction was achieved in retinal tissues obtained from live donors post-surgery. 106 5 | HUMAN RETINAL EXPLANT CULTURE CONDITIONS…”

“…Glutamine, B-27 and N2 are the most commonly used supplements in human retinal explant culture. 62,98,101,104,106 A number of growth factors like neuronal growth factor, 54 fibroblast growth factor 97 and epidermal growth factor 100 have been incorporated in the retinal culture media, specifically to enhance the survival of retinal neurons. Buffers like HEPES are used to maintain the pH of the media.…”