Sensitization of ON-bipolar cells with ambient light activatable multi-characteristic opsin rescues vision in mice

Abstract

Gene therapy-based treatment such as optogenetics offers a potentially powerful way to bypass damaged photoreceptors in retinal degenerative diseases and use the remaining retinal cells for functionalization to achieve photosensitivity. However, current approaches of optogenetic treatment rely on opsins that require high intensity light for activation thus adding to the challenge for use as part of a wearable device. Here, we report AAV2 assisted delivery of highly photosensitive multi-characteristic opsin (MCO1) into ON-bipolar cells of mice with retinal degeneration to allow activation by ambient light. Rigorous characterization of delivery efficacy by different doses of AAV2 carrying MCO1 (vMCO1) into targeted cells showed durable expression over 6 months after delivery as measured by reporter expression. The enduring MCO1 expression was correlated with the significantly improved behavioral outcome, that was longitudinally measured by visual water-maze and optomotor assays. The pro/anti-inflammatory cytokine levels in plasma and vitreous humor of the vMCO1-injected group did not change significantly from baseline or control group. Furthermore, biodistribution studies at various time points after injection in animal groups injected with different doses of vMCO1 showed non-detectable vector copies in non-targeted tissues. Immunohistochemistry of vMCO1 transfected retinal tissues showed bipolar specific expression of MCO1 and the absence of immune/inflammatory response. Furthermore, ocular imaging using SD-OCT showed no change in the structural architecture of vMCO1-injected eyes. Induction of ambient light responsiveness to remaining healthy bipolar cells in subjects with retinal degeneration will allow the retinal circuitry to gain visual acuity without requiring an active stimulation device.

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Fig. 1: Expression of ambient light activatable multi-characteristic opsin (MCO1) in retinal explant of rd10 mice led to significant photocurrent.
Fig. 2: Kinetics of vMCO1-dose dependent expression in mouse retina.
Fig. 3: Intravitreal injection of vMCO1 in rd10 mice led to ambient-light guided locomotion in a longitudinal manner.
Fig. 4: Improvement of optomotor response in vMCO1-treated rd10 mice at ambient light level.
Fig. 5: No detectable increase in inflammatory response in plasma of rd10 mice after vMCO1 injection.
Fig. 6: No detectable inflammatory response in vitreous humor or immune response in plasma of vMCO1-injected mice.
Fig. 7: Biodistribution show non-detectable levels of the vector in non-targeted organs of intravitreally-injected rd10 mice.
Fig. 8: Intravitreal injection of vMCO1 led to ON-bipolar specific expression in rd10 mice retina without causing inflammatory response.
Fig. 9: Intravitreal injection of vMCO1 did not cause cellular inflammatory response in retina of rd10 mice.

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Acknowledgements

The authors would like to thank Dr. John Repass (ARQ Genetics) for QPCR analysis; Ashutosh Tripathy and Vasu Mahapatra for their help in experiments. The authors would also like to acknowledge proofread by Dr. Darryl Narcisse (Nanoscope) and support from the National Institute of Health (1R01EY025717-01A1, 1R43EY026483-01, 1R43EY025905-01, 1R01 EY028216-01A1, and 2R44EY025905-02A1).

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SG carried out plasmid preparation, MCO1 expression analysis, in vitro/in vivo expression immunoassay, imaging, and behavioral assays; SBa performed the explant preparation, in vitro transfection, patch-clamp, and data analysis. SP carried out behavioral experiments and tissue extraction. SBh carried out behavioral data analysis and participated in discussion and planning of experiments. WW provided input on experimental design, animal model, behavioral assays, and electrophysiology. SM performed the intravitreal injections, confocal/OCT imaging, data analysis, and supervised the project. All authors contributed to the preparation of the paper.

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Correspondence to Samarendra Mohanty.

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SM and SBh have equity interest in Nanoscope Technologies, LLC, which is developing products in Biomedical diagnostics and therapeutic technologies.

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All experimental procedures were conducted according to the Nanoscope Technologies’ Institutional Animal Care and Use Committee approved protocol and standard operating procedures (SOPs).

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Batabyal, S., Gajjeraman, S., Pradhan, S. et al. Sensitization of ON-bipolar cells with ambient light activatable multi-characteristic opsin rescues vision in mice. Gene Ther (2020). https://doi.org/10.1038/s41434-020-00200-2

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