Renigunta, V. | Search bioRxiv

bioRxiv2025

Lipid Nanoparticles Enable mRNA Delivery to Diverse Cell Types of the Inner Retina

Lipid nanoparticles (LNPs) have emerged as a promising platform for retinal genetic therapy, offering a non-viral alternative to adeno-associated viruses (AAVs). While LNPs can transfect outer retinal cells, their tropism for inner retinal cell types remains insufficiently characterized. Here, we systematically assessed cellular tropism of conventional LNPs encapsulating chemically modified mRNA encoding mCherry in murine retinal explants and dissociated retinal cells. We compared quasi-subretinal and quasi-intravitreal administrations and evaluated how retinal degeneration and inner limiting membrane (ILM) integrity influence LNP-mediated transfections. We observed that LNPs efficiently transfected Muller glia under all experimental conditions. In addition, LNPs transfected several other retinal cell types, including neurons in dissociated cells and explants, and vascular cells exclusively in explants. Subretinal delivery resulted in significantly higher transfection rates than intravitreal administration, and overall efficiency was higher in degenerate as compared to non-degenerate healthy retinas. In healthy retinas, removal of ILM-associated barriers significantly increased transfection efficiency following intravitreal administration. Together, these findings demonstrate that conventional LNPs can transfect a broader range of retinal cell types than previously recognized and highlight LNPs as a versatile tool for mRNA delivery to the retina, with applications in gene supplementation, gene editing, and regenerative therapies for inner retinal disorders.

neuroscience↗

bioRxiv2024

Chloride binding and cholesterol effects on high frequency complex nonlinear capacitance (cNLC) in the mouse outer hair cell: experiment and molecular dynamics

Prestin (SLC26a5) function evolved to enhance auditory sensitivity and frequency selectivity by providing mechanical feedback via outer hair cells (OHC) into the organ of Corti. Its effectiveness is governed by the voltage-dependent kinetics of the proteins charge movements, namely, nonlinear capacitance (NLC). We study the frequency response of NLC in the mouse OHC, a species with ultrasonic hearing. We find that the characteristic frequency cut-off (Fis) for the mouse in near 27 kHz. Single point mutations within the chloride binding pocket of prestin (e.g., S396E, S398E) lack the proteins usual anion susceptibility. In agreement, we now show absence of anion binding in these mutants through molecular dynamics (MD) simulations. NLC Fis in the S396E knock-in mouse is unaltered, indicating that high frequency activity is not governed by chloride, but more likely by viscoelastic loads within the membrane. We also show that the allosteric action of chloride does not underlie piezoelectric-like behavior in prestin, since tension sensitivity of S396E NLC is comparable to that of WT. Because prestin structures of all species studied to-date are essentially indistinguishable, with analogous chloride binding pockets, auditory requirements of individual species for cochlear amplification likely evolved to enhance prestin performance by modifying, not its protein-anion interaction, but instead external mechanical loads on the protein. SignificancePrestin is believed to provide cochlear amplification in mammals that possess a wide range of frequency sensitivities. Previously, chloride anions have been shown to control prestin kinetics at frequencies below 10 kHz. However, now we find that chloride binding is not influential for prestin kinetics in the very high range of the mouse. We suggest that such high frequency prestin performance is governed by impinging mechanical loads within the membrane, and not interactions with anions.

biophysics↗