Laser‐assisted targeted gene delivery to degenerated retina improves retinal function

Abstract

Delivery of therapeutic genes into retina is proving to reverse degeneration and restore vision, however, viral vector-based gene delivery is prone to immunorejection, inflammatory/immune-response and nontargeted. Here, we report nonviral gene delivery and expression of opsin encoding genes in mouse retina in-vitro and in-vivo by use of pulsed femtosecond laser microbeam. In-vitro patch-clamp recording of the opsin-sensitized retinal cells and visually evoked in-vivo electrical recording from laser-transfected eye of mouse with degenerated retina showed functional response. The ultrafast laserbased naked gene delivery showed minimal damage and reliable expression of therapeutic opsin in cell membrane of the selected cells and in targeted retinal region. Laser-based "naked DNA gene therapy" in a spatially targeted manner will pave the way for treatment of inherited retinal diseases.

Gene-agnostic approaches to treating inherited retinal degenerations

“…In previous work, MCO1-treated mice also showed improvements in visually guided behaviors like the Morris water maze ( Wright et al, 2017 ). This was true even under illumination levels ten times lower than the thresholds typically required for channelrhodopsin stimulation in traditionally optogenetically modified mice ( Wright et al, 2017 ; Batabyal et al, 2021a ; Batabyal et al, 2021b ). Furthermore, chronic ambient light exposure for 8 h per day did not induce photobleaching in the treated mice ( Batabyal et al, 2021b ).…”

“…For some patients, cytotoxicity may eliminate AAV-mediated gene therapy as an option. A novel method for nano-enhanced optical delivery may alleviate these concerns and serve as a laser-assisted gene therapy alternative ( Batabyal et al, 2021a ). Known as optoporation, this strategy depends on a pulsed femtosecond near-infrared laser microbeam to facilitate high-efficiency, transient perforations in the cell membrane; this leads to spatially localized transfection of cells with the desired genetic material ( Matsuda and Cepko, 2004 ; Lagali et al, 2008 ; Doroudchi et al, 2011 ; Mohanty, 2012 ).…”

“…There have been notable achievements in the application of optogenetics via adeno-associated viral vectors (AAV) to restore vision to patients, while bypassing photoreceptors entirely ( Batabyal et al, 2021a ; Gauvain et al, 2021 ; Sahel et al, 2021 ). Optogenetic strategies take advantage of opsins, light-sensitive proteins often derived from bacteria, to repurpose them for scientific and medical applications.…”

Nanoactuator for Neuronal Optoporation

“…Several physical, chemical, microbic, or immune mechanisms can induce membrane damage and poration, potentially leading to cell death. In addition to these stressors, endogenously regulated cell death mechanisms involving membrane breaching by specialized pore-forming proteins or polycationic molecules exist and play an important role in tissue defense from infections and in cell turnover. , Membrane poration can also be achieved using molecular motors/switches engineered to produce nanomechanical actions on the plasma membrane triggered by adequate stimuli. , In addition to these molecular technologies, light-driven nanotechnology has also been applied to induce membrane poration with high precision and low invasiveness, leading to diverse applications from drug and gene delivery to targeted cell death. , Laser-assisted optoporation is an efficient tool for loading cells with large molecules such as cDNA or small beads, , and its throughput can be enhanced by nanosensitizers and nanostructures. − In this respect, plasmonic optoporation on three-dimensional (3D) microelectrode arrays was recently shown to open transient nanopores in cell membranes without compromising the seal between the cell membrane and the nanoelectrode …”

Vision Restoration by Optogenetic Therapy and Developments Toward Sonogenetic Therapy

“…Another approach has combined several microbial opsins (ChR2, ReaCh, and C1V1) to generate the multicharacteristic opsin (MCO1) with a white light sensitivity and apparently the need for lower light levels. 30 – 32 This approach was assessed in a dog 32 prior to the recent development of a clinical trial with the company Nanoscope Therapeutics (NCT04945772).…”