Ashutosh Tripathy

Background: Retinitis pigmentosa, a rare genetic disease, causes deterioration of light-sensing cells in the retina, which leads to night vision loss, peripheral vision, and visual sharpness. The cure for this disease poses challenges in developing gene therapies. Optogenetics offers a solution by replacing faulty rhodopsin molecules and introducing a new light-responsive pathway for photoreceptors.

Search Methods: Initial efforts focused on implanting and expressing light-gated GPCR vertebrate rhodopsin using Volvox channelrhodopsin-1 (mVChR1) but have since shifted to ReaChR and ChrimsonR variants for vision restoration applications. By constructing a plasmid containing a 4× repeat of the metabotropic glutamate receptor 6 promoter and rat rhodopsin gene tagged with yellow fluorescent protein, researchers can target ON-bipolar cells for the expression of vertebrate rhodopsin. Optogenetic actuators must be targeted to the axon and presynapse. Introducing endoplasmic reticulum (ER) export and Golgi trafficking signals from the potassium channel Kir2.1 has been shown to improve surface expression of rhodopsins. Novel methods have been developed to localize optogenetic actuators to axonal membranes, facilitating a multitude of functions.

Results: Opsin derivation and implantation methodology vary, with animal and microbial opsins being the two main types. Microbial opsins are robust but have low light sensitivity, while animal opsins offer greater light sensitivity via G protein-coupled cascades within retinal cells. Expression of human MWC opsin in the ON-bipolar cells of the retina of rd1 mice has been achieved by fusing 4xGrm6-SV40 with human rhodopsin and medium-wave opsin sequences, packaged in different AAV capsids. Limiting opsin expression to a single cell type optimizes the use of interconnecting neural pathways in the surviving retina. Medium Wavelength (MW)-Cone Opsin activation has shown promise in restoring sensitivity by rapidly activating GIRK1(F137S) G protein-coupled inward-rectifier potassium channels. Injected into blind mice, MW-Opsin displayed a significant transfection rate and enabled both static and moving pattern recognition in dim light. Its high sensitivity could eliminate the need for light-enhancing goggles in clinical trials and reduce concerns about photo-damage.

Conclusions: Expanding treatment options could lead to lower vector doses in human patients, reducing the risk of adverse intraocular inflammation and immune response. Opsin fusion proteins predominantly resemble bipolar cells, but applications could be broadened to other cell types, such as amacrine cells. Although the most efficient or long-lasting opsin implantation and expression pathways remain unknown, the proposed mechanisms show promising potential to expand applications.

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