Functional Availability of ON-Bipolar Cells in the Degenerated Retina: Timing and Longevity of an Optogenetic Gene Therapy

Abstract

Degenerative diseases of the retina are responsible for the death of photoreceptors and subsequent loss of vision in patients. Nevertheless, the inner retinal layers remain intact over an extended period of time, enabling the restoration of light sensitivity in blind retinas via the expression of optogenetic tools in the remaining retinal cells. The chimeric Opto-mGluR6 protein represents such a tool. With exclusive ON-bipolar cell expression, it combines the light-sensitive domains of melanopsin and the intracellular domains of the metabotropic glutamate receptor 6 (mGluR6), which naturally mediates light responses in these cells. Albeit vision restoration in blind mice by Opto-mGluR6 delivery was previously shown, much is left to be explored in regard to the effects of the timing of the treatment in the degenerated retina. We performed a functional evaluation of Opto-mGluR6-treated murine blind retinas using multi-electrode arrays (MEAs) and observed long-term functional preservation in the treated retinas, as well as successful therapeutical intervention in later stages of degeneration. Moreover, the treatment decreased the inherent retinal hyperactivity of the degenerated retinas to levels undistinguishable from healthy controls. Finally, we observed for the first time micro electroretinograms (mERGs) in optogenetically treated animals, corroborating the origin of Opto-mGluR6 signalling at the level of mGluR6 of ON-bipolar cells.

Bipolar cell targeted optogenetic gene therapy restores parallel retinal signaling and high-level vision in the degenerated retina

“…Notably, the visual acuity and contrast sensitivity thresholds did not differ significantly in rd1 mice that were tested at an older age (Supplementary Fig. 6) which is in line with previously reported long-term functional preservation of optogenetic gene therapies 30 . These OMR data did not directly reflect the result of Fig.…”

Selective Block of Upregulated Kv1.3 Potassium Channels in ON-Bipolar Cells of the Blind Retina Enhances Optogenetically Restored Signaling

“…The death of photoreceptors during degenerative retinal diseases, such as RP, ultimately leads to blindness [ 5 , 6 , 7 , 8 ]. OBCs, the first retinal interneurons downstream of the photoreceptors, were shown to survive for months to years after photoreceptor death [ 16 , 22 , 26 ], making them attractive targets for optogenetic vision restorative approaches [ 47 ]. Several optogenetic constructs have been introduced to OBCs to restore basic visual signaling [ 17 , 19 ].…”

“…Currently, there is only one FDA-approved gene supplementation therapy available for genetic blindness, for patients specifically carrying the biallelic mutation of the RPE65 gene [ 9 ]. Given the heterogenous genetic origin of RP, other, more universal, therapeutic approaches are being explored, amongst them electronic prostheses [ 10 , 11 ], stem cell transplantation [ 12 , 13 ], expression of photo-switchable ligands [ 14 , 15 ], and optogenetic gene therapy [ 9 , 16 , 17 ]. Optogenetic gene therapy was shown to restore some visual function in a human patient [ 18 ] and in murine models of RP, particularly when targeted to the OBCs [ 19 , 20 , 21 , 22 ] such as Opto-mGluR6 [ 20 , 22 ].…”

CRISPR-mediated optogene expression from a cell-specific endogenous promoter in retinal ON-bipolar cells to restore vision

“…Opto-mGluR6, a chimera of proteins of human origin, is about 500 times more light sensitive than ChR2 and naturally triggers endogenous TRPM1 signaling in ON-BPCs (van Wyk et al, 2015). We have previously demonstrated that the intravitreal injection of AAVs carrying the Opto-mGluR6 optogene under the control of the ON-BPC-specific GRM6 promoter (Hulliger et al, 2020) can restore visual function in otherwise blind retinal degeneration (rd1) mice (van Wyk et al, 2015;Kralik and Kleinlogel, 2021).…”

“…For AAV vector generation, we used an AAV backbone with AAV2 ITRs while, for non-AAV vectors, we used the pIRES backbone. For details of vector composition, see (van Wyk et al, 2017;Hulliger et al, 2020;Kralik and Kleinlogel, 2021). The miniCMV promoter was derived from plasmid pX551-miniCMV-SpCas9 (Addgene plasmid # 107031).…”