Assessment of Adeno-Associated Virus Serotype Tropism in Human Retinal Explants

Abstract

Advances in the discovery of the causes of monogenic retinal disorders, combined with technologies for the delivery of DNA to the retina, offer enormous opportunities for the treatment of previously untreatable blinding diseases. However, for gene augmentation to be most effective, vectors that have the correct cell-type specificity are needed. While animal models are very useful, they often exhibit differences in retinal cell surface receptors compared to the human retina. This study evaluated the use of an ex vivo organotypic explant system to test the transduction efficiency and tropism of seven different adeno-associated virus type 2 (AAV2) serotypes in the human retina and retinal pigment epithelium-choroid-AAV2/1, AAV2/2, AAV2/4, AAV2/5, AAV2/6, AAV2/8, and AAV2/9-all driving expression of GFP under control of the cytomegalovirus promoter. After 7 days in culture, it was found that AAV2/4 and AAV2/5 were particularly efficient at transducing photoreceptor cells and that AAV2/5 was highly specific to the outer nuclear layer, whereas AAV2/8 displayed consistently low transduction of photoreceptors. To validate the authenticity of the organotypic culture system, the transduction of the same set of AAVs was also compared in a pig model, in which sub-retinal injections in vivo were compared to cultured and transduced organotypic cultures ex vivo. This study shows how different AAV serotypes behave in the human retina and provides insight for further investigation of each of these serotypes for gene augmentation-based treatment of inherited retinal degeneration.

AAV-Mediated Gene Therapy for CRB1-Hereditary Retinopathies

“…Therefore, is quite difficult to extrapolate the data from pre-clinical studies performed in rodents directly to the human in vivo setting. To obtain evidence-based data for clinical gene therapy studies, researchers optimize culture protocols for human retinal organotypic cultures [77][78][79] or human iPSC-derived retinas to study the AAV tropism [80]. Recently, the capacity of different AAV serotypes to infect and express in human retinal cells was studied in organotypic…”

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

“…Although Fradot et al reported lack of AAV transduction in human retinal explant culture in 2011, researchers were able to successfully transduce and reactivate human RGCs in retinal explant culture using both lentiviral and AAV vectors in the later years . However, Wiley et al upon exploring AAV serotype tropism observed differences in the transduction efficiency between human and porcine retinal explant models . 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 .…”

“…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.…”

“…103 However, Wiley et al upon exploring AAV serotype tropism observed differences in the transduction efficiency between human and porcine retinal explant models. 104 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. 105 Despite these differences, AAV-mediated viral transduction was achieved in retinal tissues obtained from live donors post-surgery.…”

“…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.…”