A universal protocol for isolating retinal ON bipolar cells across species via fluorescence-activated cell sorting

Elisa Murenu,Marina Pavlou,Lisa Richter,Kleopatra Rapti,Sabrina Just,Jasmina Cehajic-Kapetanovic,Neda Tafrishi,Andrew Hayes,Rachel Scholey,Robert Lucas,Hildegard Büning,Dirk Grimm,Stylianos Michalakis

doi:10.1016/j.omtm.2021.01.011

Abstract

Inherited retinal dystrophies (IRDs) are characterized by progressive degeneration and loss of light-sensing photoreceptors. The most promising therapeutic approach for IRDs is gene supplementation therapy using viral vectors, which requires the presence of viable photoreceptors at the time of intervention. At later disease stages, photoreceptors are lost and can no longer be rescued with this approach. For these patients, conferring light-sensing abilities to the remaining interneurons of the ON circuit (i.e., ON bipolar cells) using optogenetic tools poses an alternative treatment strategy. Such treatments, however, are hampered by the lack of efficient gene delivery tools targeting ON bipolar cells, which in turn rely on the effective isolation of these cells to facilitate tool development. Herein, we describe a method to selectively isolate ON bipolar cells via fluorescence-activated cell sorting (FACS), based on the expression of two intracellular markers. We show that the method is compatible with highly sensitive downstream analyses and suitable for the isolation of ON bipolar cells from healthy as well as degenerated mouse retinas. Moreover, we demonstrate that this approach works effectively using non-human primate (NHP) retinal tissue, thereby offering a reliable pipeline for universal screening strategies that do not require inter-species adaptations or transgenic animals.

Highlights

Inherited retinal dystrophies (IRDs) are a major cause of blindness worldwide
ON bipolar cells are better suited targets for optogenetic therapies, as signals generated in these neurons are further processed within the retina at synapses in the inner plexiform layer (IPL)[8] before they are sent to the thalamus by the ganglion cells
Goa and Pcp[2] label ON bipolar cells in healthy and degenerated mouse retinas In order to develop a protocol for sorting ON bipolar cells, cytoplasmic markers were assessed for their specific and stable expression in this cell population

Summary

Introduction

Inherited retinal dystrophies (IRDs) are a major cause of blindness worldwide. Among the approaches developed to alleviate this condition, gene supplementation therapy represents to date the most clinically relevant. (legend on page) www.moleculartherapy.org darkness.[7,8] While ganglion cells are suitable candidates due to their accessible location, their position at the end of the retinal circuitry does not allow for any signal amplification or fine-tuning that is required for daylight vision. In this regard, ON bipolar cells are better suited targets for optogenetic therapies, as signals generated in these neurons are further processed within the retina at synapses in the inner plexiform layer (IPL)[8] before they are sent to the thalamus by the ganglion cells. Efficient vectors and tools that enable specific targeting of ON bipolar cells across species are still missing

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Conclusion