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Dysfunctions caused by gene mutations in the retinal pigment epithelium (RPE) lead to retinal degeneration, visual function loss, and even blindness. RPE-specific gene replacement therapy holds great promise for treating monogenic ocular disorders in the RPE. Although the adeno-associated virus (AAV) has been approved for gene therapy to treat monogenic visual disorders, broad clinical applications of AAV-based gene therapy are limited by its gene loading capacity. In this work, we intended to design and develop a stable retinylamine analogue ACU4429-modified dual pH-sensitive ECO/pDNA nanoparticles for specific delivery of large therapeutic genes to the RPE. ACU4429 was first conjugated to a PEG 3.4 kD spacer with a pH-sensitive hydrazone linker at the distal end (ACU-PEG-HZ-MAL). The targeted dual pH-sensitive ECO/pDNA nanoparticles were then prepared by self-assembly of ACU-PEG-HZ-MAL, pH-sensitive lipid carrier ECO, and a plasmid DNA expressing the large ABCA4 gene. The formation of targeted ACU-PEG-HZ-ECO/pDNA nanoparticles was characterized by dynamic light scattering and gel electrophoresis. The incorporation of a hydrazone linker enhanced the cytosolic gene delivery, which translated to high ABCA4 expression in ARPE-19 cells for ACU-PEG-HZ-ECO/pABCA4 nanoparticles. The targeted nanoparticles also demonstrated excellent targeting efficiency in the interphotoreceptor matrix of Abca4-/- mice, resulting in enhanced expression of the ABCA4 gene in the RPE. The ACU4429 PEG hydrazone-modified ECO/pDNA nanoparticles provide a promising nonviral platform to safely and effectively deliver therapeutic genes with unlimited sizes for the treatment of monogenic visual disorders.

Stargardt disease (STGD) is an autosomal recessive retinal disorder caused by a monogenic ABCA4 mutation. Currently, there is no effective therapy to cure Stargardt disease. The replacement of mutated ABCA4 with a functional gene remains an attractive strategy. In this study, we have developed a non-viral gene therapy using nanoparticles self-assembled by a multifunctional pH-sensitive amino lipid ECO and a therapeutic ABCA4 plasmid. The nanoparticles mediated efficient intracellular gene transduction in wild-type (WT) and Abca4-/- mice. Specific ABCA4 expression in the outer segment of photoreceptors was achieved by incorporating a rhodopsin promoter into the plasmids. The ECO/pRHO-ABCA4 nanoparticles induced substantial and specific ABCA4 expression for at least 8 months, 35% reduction in A2E accumulation on average, and a delayed Stargardt disease progression for at least 6 months in Abca4-/- mice. ECO/plasmid nanoparticles constitute a promising non-viral gene therapy platform for Stargardt disease and other visual dystrophies.

Noninvasive imaging of visual system components in vivo is critical for understanding the causal mechanisms of retinal diseases and for developing therapies for their treatment. However, ultraviolet light needed to excite endogenous fluorophores that participate in metabolic processes of the retina is highly attenuated by the anterior segment of the human eye. In contrast, 2-photon excitation fluorescence imaging with pulsed infrared light overcomes this obstacle. Reducing retinal exposure to laser radiation remains a major barrier in advancing this technology to studies in humans. To increase fluorescence intensity and reduce the requisite laser power, we modulated ultrashort laser pulses with high-order dispersion compensation and applied sensorless adaptive optics and custom image recovery software and observed an over 300% increase in fluorescence of endogenous retinal fluorophores when laser pulses were shortened from 75 fs to 20 fs. No functional or structural changes to the retina were detected after exposure to 2-photon excitation imaging light with 20-fs pulses. Moreover, wide bandwidth associated with short pulses enables excitation of multiple fluorophores with different absorption spectra and thus can provide information about their relative changes and intracellular distribution. These data constitute a substantial advancement for safe 2-photon fluorescence imaging of the human eye.

Cellular uptake of vitamin A, production of visual chromophore and triglyceride homeostasis in adipocytes depend on two representatives of the vertebrate N1pC/P60 protein family, lecithin:retinol acyltransferase (LRAT) and HRAS-like tumor suppressor 3 (HRASLS3). Both proteins function as lipid-metabolizing enzymes but differ in their substrate preferences and dominant catalytic activity. The mechanism of this catalytic diversity is not understood. Here, by using a gain-of-function approach, we identified a specific sequence responsible for the substrate specificity of N1pC/P60 proteins. A 2.2-Å crystal structure of the HRASLS3-LRAT chimeric enzyme in a thioester catalytic intermediate state revealed a major structural rearrangement accompanied by three-dimensional domain swapping dimerization not observed in native HRASLS proteins. Structural changes affecting the active site environment contributed to slower hydrolysis of the catalytic intermediate, supporting efficient acyl transfer. These findings reveal structural adaptation that facilitates selective catalysis and mechanism responsible for diverse substrate specificity within the LRAT-like enzyme family. [ABSTRACT FROM AUTHOR]