Non-viral gene therapy for Leber's congenital amaurosis: progress and possibilities

Abstract

Leber's congenital amaurosis (LCA) represents a set of rare and pervasive hereditary conditions of the retina that cause severe vision loss starting in early childhood. Targeted treatment intervention has become possible thanks to recent advances in understanding LCA genetic basis. While viral vectors have shown efficacy in gene delivery, they present challenges related to safety, low cargo capacity, and the potential for random genomic integration. Non-viral gene therapy is a safer and more flexible alternative to treating the underlying genetic mutation causing LCA. Non-viral gene delivery methods, such as inorganic nanoparticles, polymer-based delivery systems, and lipid-based nanoparticles, bypass the risks of immunogenicity and genomic integration, potentially offering a more versatile and personalized treatment for patients. This review explores the genetic background of LCA, emphasizing the mutations involved, and explores diverse non-viral gene delivery methods being developed. It also highlights recent studies on non-viral gene therapy for LCA in animal models and clinical trials. It presents future perspectives for gene therapy, including integrating emerging technologies like CRISPR-Cas9, interdisciplinary collaborations, personalized medicine, and ethical considerations.

Keywords: DNA nanoparticle; Leber’s congenital amaurosis; degeneration; gene therapy; genetics; non-viral vectors; ocular gene therapy.

Figure 1.

An overview of the etiology of LCA caused by visual cycle disruption (1) absorption of light by the photoreceptor cells initiates the commencement of the visual cycle. (2) when photons are absorbed, 11-cis-retinal transforms into all-trans-retinal, causing a conformational change in the photoreceptor cells. This conformational change activates a signaling pathway known as the phototransduction cascade, which involves the reduction of all-trans-retinal into all-trans-retinol. this is then transported to the retinal pigment epithelium (RPE). In the RPE, lecithin retinol acyltransferase (LRAT) converts all-trans-retinol to retinyl esters. Retinyl esters are converted into 11-cis-retinol by the enzyme RPE65, also known as retinoid isomerohydrolase. 11-cis-retinol is oxidized by retinol dehydrogenase 5 (RDH5) into 11-cis-retinal, which then diffuses into the photoreceptor segment of the retina to start the cycle again. Mutations in the RPE65 gene, can lead to dysfunctions and clinical manifestations that contribute to the development of Leber congenital amaurosis (LCA). This diagram was made with Biorender.com (assessed on 2 May 2024).

Figure 2.

LCA associated with a mutation in the GUCY2D gene. GUCY2D encodes the enzyme retinal guanylate cyclase 1 (RetGC1), which is essential in the phototransduction pathway in retinal cells by converting guanosine triphosphate (GTP) into cyclic guanosine monophosphate (cGMP). When photoreceptor cells are exposed to light, the light-sensitive pigments in the outer segments of these cells undergo a structural change. This change triggers a series of events that lead to the breakdown of cGMP. As a result, ion channels close, causing the cells to become hyperpolarized. After light exposure, the level of cGMP must be restored to maintain the normal photocurrent in the photoreceptor cells. Mutation in the GUCY2D gene impairs the function of RetGC1, leading to insufficient production of cGMP necessary for maintaining the phototransduction cycle. This diagram was made with Biorender.com (assessed on 13 November 2024).

Figure 3.

Schematic representation of the mechanism of QR-110. (1) wild-type CEP290 gene results in the production of functional CEP290 protein essential for the growth and uptake of the connecting cilium in the retinal photoreceptor cells. (2) a strong splice donor site produced by a mutation in the CEP290 gene results in the introduction of a cryptic exon (exon X) in CEP290 mRNA. An inactive, truncated CEP290 protein or a nonsense-mediated decay of the mutated mRNA transcript significantly lowers the level of wild-type CEP290 protein. (3) upon treatment with QR-110 oligonucleotide, aberrant splicing would be blocked. Wild-type transcript and CEP290 protein levels rise as a result. This diagram was made with Biorender.com (assessed on 15 November 2024).

Figure 4.

Mechanism of all-trans-retinylamine-modified ECO nanoparticles. When injected into the subretinal space, ECO nanoparticles attach to interphotoreceptor retinol-binding protein (IRBP) within the interphotoreceptor matrix. Upon entering the cells, the nanoparticle escapes the endosomal compartment and releases the RPE65 plasmid DNA. The RPE65 gene is expressed in RPE cells, which helps slow down cone cell degeneration and preserve visual function. Adapted from “targeted multifunctional lipid ECO plasmid DNA nanoparticles as efficient non-viral gene therapy for Leber’s congenital amaurosis” by Da Sun et al., 2017. This diagram was created using Biorender.com (assessed on 13 November 2024).

Figure 5.

Non-viral Gene Delivery Systems can be used to deliver genes, including DNA, mRNA, and large transgenes such as base editors, to the retina. (1) a few examples of non-viral vectors. Strategies of ocular Gene therapy: (2) the genes are released when the nanoparticles leave the endosomal compartment after being taken up by the cell through endocytosis. DNA encoding the CRISP/Cas9 system is first transported to the nucleus (except in cases where it is delivered as mRNA) and translated into Cas9 mRNA and gRNA. (3) in contrast, miRNA needs to be loaded into the RNA-induced Silencing Complex (RISC) and bind to mRNA in the cytoplasm. This diagram was made with Biorender.com (assessed on 24 April 2024).