In Vitro Gene Delivery in Retinal Pigment Epithelium Cells by Plasmid DNA-Wrapped Gold Nanoparticles.

Trigueros Trigueros,Marfany Marfany,Toulis Toulis,Domènech Domènech

doi:10.3390/genes10040289

Abstract

Many rare diseases course with affectation of neurosensory organs. Among them, the neuroepithelial retina is very vulnerable due to constant light/oxidative stress, but it is also the most accessible and amenable to gene manipulation. Currently, gene addition therapies targeting retinal tissue (either photoreceptors or the retinal pigment epithelium), as a therapy for inherited retinal dystrophies, use adeno-associated virus (AAV)-based approaches. However, efficiency and safety of therapeutic strategies are relevant issues that are not always resolved in virus-based gene delivery and alternative methodologies should be explored. Based on our experience, we are currently assessing the novel physical properties at the nanoscale of inorganic gold nanoparticles for delivering genes to the retinal pigment epithelium (RPE) as a safe and efficient alternative approach. In this work, we present our preliminary results using DNA-wrapped gold nanoparticles (DNA-gold NPs) for successful in vitro gene delivery on human retinal pigment epithelium cell cultures, as a proof-of-principle to assess its feasibility for retina in vivo gene delivery. Our results show faster expression of a reporter gene in cells transfected with DNA-gold NPs compared to DNA-liposome complexes. Furthermore, we show that the DNA-gold NPs follow different uptake, internalization and intracellular vesicle trafficking routes compared to pristine NPs.

Highlights

The dysfunction and death of photoreceptors are the main cause of vision loss in inherited retinal diseases (IRDs), a group of mendelian rare disorders with high genetic and clinical heterogeneity.After more than 30 years of intense clinical and genetic studies to identify IRD genes, around300 causative genes have been identified that cause the dysfunction of photoreceptors or alter the function of the adjacent retinal pigment epithelium (RPE), leading to the progressive attrition of photoreceptors [1,2].From the clinical side and most relevant to patients, the main challenge is to devise effective treatments to halt the progression of the disease or regain visual capacity
In IRDs, retinal degeneration is caused by mutations in either genes expressed in photoreceptors or genes expressed in the adjacent RPE, as it is the case with mutations in RPE65, MERTK or LRAT
associated virus (AAV)-based vectors are being used for gene delivery to photoreceptors, but non-viral vectors based on NPs should be concurrently explored for cases in which viral approaches may not be desirable [3,4,8]

Summary

Introduction

The dysfunction and death of photoreceptors are the main cause of vision loss in inherited retinal diseases (IRDs), a group of mendelian rare disorders with high genetic and clinical heterogeneity.After more than 30 years of intense clinical and genetic studies to identify IRD genes, around300 causative genes have been identified that cause the dysfunction of photoreceptors or alter the function of the adjacent retinal pigment epithelium (RPE), leading to the progressive attrition of photoreceptors [1,2].From the clinical side and most relevant to patients, the main challenge is to devise effective treatments to halt the progression of the disease or regain visual capacity. The dysfunction and death of photoreceptors are the main cause of vision loss in inherited retinal diseases (IRDs), a group of mendelian rare disorders with high genetic and clinical heterogeneity. Ocular gene therapy for retinal disorders is starting to be developed and the first commercial gene therapy to treat a severe infantile genetic blindness (Leber congenital amaurosis caused by bi-allelic mutations in the RPE65 gene) was approved last year. These and other ongoing therapies in different clinical trial phases are based on adeno-associated virus (AAV) vectors [6]. Viral gene delivery raises several concerns, such as small size capacity (maximum packaging size for 5 kb), high production costs, probability of immunogenicity and inflammatory responses and their invasive route of administration to the retina (subretinal microinjection), which have fostered basic research on non-viral vectors such as nanoparticles (NPs) for gene delivery

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