Periodic Reporting for period 4 - HybridRetina (Hybrid Retinal Prosthesis: High-Resolution Electrode Array Integrated with Neurons for Restoration of Sight)
Reporting period: 2022-11-01 to 2024-04-30
Degenerative diseases of the outer retina such as Retinitis Pigmentosa (RP) and Age-related Macular Degeneration (AMD) are the main underlying causes of blindness. In these diseases “the image capturing layer” of the retina, the photoreceptors, degenerate whereas the remaining neural cells that process the information (bipolar and horizontal cells) and relay it to the brain (the ganglion cells) are left relatively intact. Most current strategies for vision restoration rely on the electrical stimulation of these remaining cells. While these approaches offer promising improvements in the life quality of the transplanted patients, the obtained visual acuity is far from the desired normal visual acuity. Alternative approaches rely on novel advancements in stem cell research and are based on cellular replacement therapy. In this approach photoreceptors are differentiated from either stem cells (ES) or induced pluripotent stem cells (iPSC) and are transplanted in the subretinal space to replace the degenerate photoreceptors. Notwithstanding the demonstration of vision restoration using this approach in several animal models, it is still limited by the need for obtaining fully mature photoreceptors and the proper integration of the transplanted cells with the host retina.
To overcome these limitations, our group pursued a novel approach, which we term the hybrid retina (HRI), relying on the incorporation of differentiated photoreceptors with a high-density electrode implant and its implantation in the subretinal space. We hypothesized that the transplanted cells will properly integrate with the host retina and that their electrical stimulation through the electrodes on which they are seeded will result in a retinal prosthesis providing unprecedented visual acuity. Throughout the project we have made great strides towards the realization of this challenging approach. Whereby we have successfully fabricated a high-density electrode array device at the bottom of each well is a gold electrode centrally located for neuronal electrical stimulation. Moreover, we have demonstrated the integration of this device incorporated with photoreceptor precursors with the host retina of a blind rat model. Furthermore, we have identified optimal electrode surface treatments which enhance the cell-electrode coupling, thus reducing activation thresholds and increasing the spatial resolution of the obtained prosthetic vision. Furthermore, we identified small molecules that putatively improve the integration of the transplanted cells with the host retina and promote synaptogenesis. Finally, using an in-vitro prototype of the implant we demonstrated the significant reduction of activation thresholds of cells seeded on the device, compared with cells seeded on planar flat electrodes.
The Hybrid Retinal Implant, thus, introduces a promising solution to a large population of AMD patients who would only benefit from a high-resolution vision restoration, as their peripheral vision is largely still intact.
To overcome these limitations, our group pursued a novel approach, which we term the hybrid retina (HRI), relying on the incorporation of differentiated photoreceptors with a high-density electrode implant and its implantation in the subretinal space. We hypothesized that the transplanted cells will properly integrate with the host retina and that their electrical stimulation through the electrodes on which they are seeded will result in a retinal prosthesis providing unprecedented visual acuity. Throughout the project we have made great strides towards the realization of this challenging approach. Whereby we have successfully fabricated a high-density electrode array device at the bottom of each well is a gold electrode centrally located for neuronal electrical stimulation. Moreover, we have demonstrated the integration of this device incorporated with photoreceptor precursors with the host retina of a blind rat model. Furthermore, we have identified optimal electrode surface treatments which enhance the cell-electrode coupling, thus reducing activation thresholds and increasing the spatial resolution of the obtained prosthetic vision. Furthermore, we identified small molecules that putatively improve the integration of the transplanted cells with the host retina and promote synaptogenesis. Finally, using an in-vitro prototype of the implant we demonstrated the significant reduction of activation thresholds of cells seeded on the device, compared with cells seeded on planar flat electrodes.
The Hybrid Retinal Implant, thus, introduces a promising solution to a large population of AMD patients who would only benefit from a high-resolution vision restoration, as their peripheral vision is largely still intact.
To realize the HRI concept, the main project was separated to several aims. The first aim was the fabrication of a high density multielectrode array embedded at the bottom of insulating 3D microwell- like structures. This has been achieved through a multistep 3D lithography fabrication process which we have optimized and recently published (Shpun et al 2023). The process yielded in a 1mm diameter implant, with 3,172 wells, 10µm pixels diameter and a pixel pitch of 13µm. Next, we addressed the cell source choice and developed an optimized protocol for the differentiation of hESc into photoreceptor precursors, yielding in a highly (~80%) efficient differentiation (Markus et al 2019).As the obtained photoreceptor precursors are novel, with little known of their biophysical behavior we set at characterizing their electrophysiological characterization (patch clamp) and analysis of the expression level of several genes of interest throughout maturation (Shick et al 2020) .Our investigations revealed that these cells expressed all the necessary machinery required for membrane potential modulation as evident from the gene expression and the presence of voltage gated ion channels.
The next step in the realization of the HRI concept is enhancing the cell-electrode coupling to reduce the activation threshold. Enhancing cell-electrode coupling through biomimetic molecules was explored; our work identified the biomimetic molecule YIGSR as the optimal surface treatment for retinal cells (Shpun et al 2024). This discovery has a potential for improving the cell coupling of neurons to electrodes in the hybrid implant and in conventional retinal prostheses. In addition, we performed animal studies where devices seeded with cells were implanted in the subretinal space of a well-established model of outer retinal degeneration in rats. Using advanced imaging and histology we found that cells survived for up to 30-days and correctly localized in the subretinal space. Immunohistochemistry study of the isolated retinas revealed putative synapses between the transplanted cells and the host retina. Integration of the cells being a key factor in the successful realization of our novel concept, we set at further promoting neurite extension using growth factors and small molecules. We found significant neurite extension in photoreceptor precursors following treatment with ROCK inhibitors, Taurine and retinal culture medium (enriched with factors secreted by Muller cells of the retina) potentially increasing synaptic formation between the hybrid implant and the host retina (paper under review). These are promising results, but more investigation is still underway to better understand the nature of the formed synapses and improve the survival of the cells.
Finally, computer modeling was performed with results revealing the strong dependence of the activation threshold on the sealing (particularly the space between the cell membrane and microwell) and the reduction of the activation threshold by 4 orders of magnitude when comparing to a conventional electrode. These simulation results were validated in an in-vitro prototype. The results are now under preparation for publication.
Furthermore, we established a novel animal model, the GCaMP6-RCS rat, which expresses the calcium indicator in the RGCs, enabling the visualization of RGCs responses upon electrical stimulation (Azrad-Leibovitch et al 2024). We are now using this model to study the full implant function both in in-vivo and ex-vivo setups to fully characterize the performance of the hybrid implant.
The next step in the realization of the HRI concept is enhancing the cell-electrode coupling to reduce the activation threshold. Enhancing cell-electrode coupling through biomimetic molecules was explored; our work identified the biomimetic molecule YIGSR as the optimal surface treatment for retinal cells (Shpun et al 2024). This discovery has a potential for improving the cell coupling of neurons to electrodes in the hybrid implant and in conventional retinal prostheses. In addition, we performed animal studies where devices seeded with cells were implanted in the subretinal space of a well-established model of outer retinal degeneration in rats. Using advanced imaging and histology we found that cells survived for up to 30-days and correctly localized in the subretinal space. Immunohistochemistry study of the isolated retinas revealed putative synapses between the transplanted cells and the host retina. Integration of the cells being a key factor in the successful realization of our novel concept, we set at further promoting neurite extension using growth factors and small molecules. We found significant neurite extension in photoreceptor precursors following treatment with ROCK inhibitors, Taurine and retinal culture medium (enriched with factors secreted by Muller cells of the retina) potentially increasing synaptic formation between the hybrid implant and the host retina (paper under review). These are promising results, but more investigation is still underway to better understand the nature of the formed synapses and improve the survival of the cells.
Finally, computer modeling was performed with results revealing the strong dependence of the activation threshold on the sealing (particularly the space between the cell membrane and microwell) and the reduction of the activation threshold by 4 orders of magnitude when comparing to a conventional electrode. These simulation results were validated in an in-vitro prototype. The results are now under preparation for publication.
Furthermore, we established a novel animal model, the GCaMP6-RCS rat, which expresses the calcium indicator in the RGCs, enabling the visualization of RGCs responses upon electrical stimulation (Azrad-Leibovitch et al 2024). We are now using this model to study the full implant function both in in-vivo and ex-vivo setups to fully characterize the performance of the hybrid implant.
We believe that the novel presented concept will be able to overcome the main limitations of currently available prostheses. Specifically, it will offer restoration of sight at an unprecedented visual acuity, enable the selective activation of retinal circuitry and facilitate an analogue mode of operation. All leading to a restored prosthetic vision comparable to that of the natural one.