Recombinant adeno-associated viral vectors (AAVs) are an effective system for gene transfer. AAV serotype 2 (AAV2) is commonly used to deliver transgenes to retinal ganglion cells (RGCs) via intravitreal injection. The AAV serotype however is not the only factor contributing to the effectiveness of gene therapies. Promoters influence the strength and cell-selectivity of transgene expression. This study compares five promoters designed to maximise AAV2 cargo space for gene delivery: chicken β-actin (CBA), cytomegalovirus (CMV), short CMV early enhancer/chicken β-actin/short β-globulin intron (sCAG), mouse phosphoglycerate kinase (PGK), and human synapsin (SYN). The promoters driving enhanced green fluorescent protein (eGFP) were examined in adult C57BL/6J mice eyes and tissues of the visual system. eGFP expression was strongest in the retina, optic nerves and brain when driven by the sCAG and SYN promoters. CBA, CMV, and PGK had moderate expression by comparison. The SYN promoter had almost exclusive transgene expression in RGCs. The PGK promoter had predominant expression in both RGCs and AII amacrine cells. The ubiquitous CBA, CMV, and sCAG promoters expressed eGFP in a variety of cell types across multiple retinal layers including Müller glia and astrocytes. We also found that these promoters could transduce human retina ex vivo, although expression was predominantly in glial cells due to low RGC viability. Taken together, this promoter comparison study contributes to optimising AAV-mediated transduction in the retina, and could be valuable for research in ocular disorders, particularly those with large or complex genetic cargos.

Cambridge Institute for Medical Research University of Cambridge Cambridge CB2 0XY UK

Centre for Eye Research Australia Royal Victorian Eye and Ear Hospital Melbourne VIC Australia

Centre for Neurogenomics and Cognitive Research Amsterdam Neuroscience Vrije Universiteit Amsterdam Amsterdam The Netherlands

Centre of Reconstructive Neuroscience Institute of Experimental Medicine Prague Czech Republic

Department of Clinical Neuroscience Division of Eye and Vision St Erik Eye Hospital Karolinska Institutet Stockholm Sweden

Ikarovec Ltd The Norwich Research Park Innovation Centre Norwich UK

John van Geest Centre for Brain Repair Department of Clinical Neurosciences University of Cambridge Cambridge UK

Laboratory for Regeneration of Sensorimotor Systems Netherlands Institute for Neuroscience Royal Netherlands Academy of Arts and Sciences Amsterdam The Netherlands

Ophthalmology Department of Surgery University of Melbourne Melbourne VIC Australia

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