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. 2022 Jul 13;33(13-14):695–707. doi: 10.1089/hum.2021.272

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© Tiffany S. Nelson et al., 2022. Published by Mary Ann Liebert, Inc.

This Open Access article is distributed under the terms of the Creative Commons Attribution Noncommercial License [CC-BY-NC] (http://creativecommons.org/licenses/by-nc/4.0/) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and the source are cited.

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Figure 2. — Safety and efficacy of ArrGG overexpression in adult C57BL/6J mouse retina. (A) Design of AAV vectors with myc-tagged modified arrestin1 (ArrGG) and wild-type arrestin1 (ArrWT) transgene expression driven by a human rod-specific opsin promoter (hOp181opt). The expression cassette is flanked by ITR of AAV serotype 2. (B) ERG responses at 60 days postinjection; example raw scotopic and photopic responses from an animal injected with ArrGG (green) compared with one injected with buffer (gray) are shown above; flash intensity response curves for scotopic and photopic amplitudes were not significantly different in eyes injected with ArrGG (n = 8) compared with eyes injected with just buffer (n = 4); points represent mean ± SEM. (C) Example of ONL thickness measured by OCT in mouse injected with buffer (left image) or ArrGG (right image; scale bars, 50 μm); spider gram plot of ONL thickness measured in eyes treated with ArrGG (green curves; n = 8) shows no significant different compared with eyes receiving buffer only (black curves; n = 4); points represent mean ± SEM. (D) Lactate secretion rates in retinal explants from mice treated with ArrGG (green; n = 8) and mice receiving buffer only (gray; n = 4); bars show mean ± SEM, asterisk indicates statistically significant difference in parametric paired t-test (ρ < 0.05). (E) Immunoblot quantitation of ArrGG expression relative to endogenous arrestin1; blots of soluble extracts of retinas used in (D) from seven eyes injected with AAV-ArrGG and two eyes injected with buffer (indicated below blot) were probed with anti-arrestin1 antibody, which identifies the higher molecular mass ArrGG (with a myc-tag) and the native arrestin1; values above each lane indicate the amount of ArrGG expressed as a percentage of the native arrestin1. (F) Immunohistochemistry of representative mouse retinal sections; paraffin sections of eyes injected with AAV-ArrGG or buffer were stained for arrestin1 (green) or for myc antigen (red), and nuclei stained with DAPI (blue). Scale bars, 10 μm. ERG, electroretinograms; GCL, ganglion cell layer; INL, inner nuclear layer; IS/OS, inner segment/outer segment layers; OCT, optical coherence tomography; ONL, outer nuclear layer; RPE, retinal pigmented epithelium. Color images are available online.