Outer Plexiform Layer Structures Are Not Altered Following AAV-Mediated Gene Transfer in Healthy Rat Retina.

Bert C Giers,Carolina Isiegas,Daniela Klein,Alexandra Mendes-Madeira,Knut Stieger,Birgit Lorenz,Silke Haverkamp

doi:10.3389/fneur.2017.00059

Abstract

Ocular gene therapy approaches have been developed for a variety of different diseases. In particular, clinical gene therapy trials for RPE65 mutations, X-linked retinoschisis, and choroideremia have been conducted at different centers in recent years, showing that adeno-associated virus (AAV)-mediated gene therapy is safe, but limitations exist as to the therapeutic benefit and long-term duration of the treatment. The technique of vector delivery to retinal cells relies on subretinal injection of the vector solution, causing a transient retinal detachment. Although retinal detachments are known to cause remodeling of retinal neuronal structures as well as significant cell loss, the possible effects of this short-term therapeutic retinal detachment on retinal structure and circuitry have not yet been studied in detail. In this study, retinal morphology and apoptotic status were examined in healthy rat retinas following AAV-mediated gene transfer via subretinal injection with AAV2/5.CMV.d2GFP or sham injection with fluorescein. Outer plexiform layer (OPL) morphology was assessed by immunohistochemical labeling, laser scanning confocal microscopy, and electron microscopy. The number of synaptic contacts in the OPL was quantified after labeling with structural markers. To assess the apoptotic status, inflammatory and pro-apoptotic markers were tested and TUNEL assay for the detection of apoptotic nuclei was performed. Pre- and postsynaptic structures in the OPL, such as synaptic ribbons or horizontal and bipolar cell processes, did not differ in size or shape in injected versus non-injected areas and control retinas. Absolute numbers of synaptic ribbons were not altered. No signs of relevant gliosis were detected. TUNEL labeling of retinal cells did not vary between injected and non-injected areas, and apoptosis-inducing factor was not delocalized to the nucleus in transduced areas. The neuronal circuits in the OPL of healthy rat retinas undergoing AAV-mediated gene transfer were not altered by the temporary retinal detachment caused by subretinal injection, the presence of viral particles, or the expression of green fluorescent protein as a transgene. This observation likely requires further investigations in the dog model for RPE65 deficiency in order to determine the impact of RPE65 transgene expression on diseased retinas in animals and men.

Highlights

Adeno-associated virus (AAV)-mediated gene therapy has facilitated tremendous progress in the therapy of hereditary retinal dystrophies in the last decade
The AAV2/5 vector used in this study is known to transduce with high-efficiency photoreceptors and RPE cells following subretinal injection
We show that subretinal delivery of AAV vectors does not cause significant alterations to structures in the Outer plexiform layer (OPL) in terms of qualitative features, such as increased sprouting of bipolar cells or synaptic ribbon structures, or quantitative features, such as the absolute number of synaptic terminals

Summary

Introduction

Adeno-associated virus (AAV)-mediated gene therapy has facilitated tremendous progress in the therapy of hereditary retinal dystrophies in the last decade. A growing number of genetic mutations have been identified over the past decades that can cause retinal dystrophy [3], among which mutations in the gene encoding the retinal pigment epithelial protein of 65 kDa (RPE65) are best characterized [4] This isomerase plays a crucial role in the visual cycle restoring the light sensitive chromophore 11-cis retinal from all-trans retinal [5, 6]. Several animal models have been described to model the pathological findings of RPE65 deficiency in human patients, most importantly the naturally occurring canine model of RPE65 deficiency, the Swedish Briard dog [8] All of these animal models have been successfully used to implement new therapeutic strategies, showing that AAV-mediated gene therapy is able to restore significant vision in affected animals [9,10,11,12,13]

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