Harnessing the Potential of Human Pluripotent Stem Cells and Gene Editing for the Treatment of Retinal Degeneration

Abstract

Purpose of review: A major cause of visual disorders is dysfunction and/or loss of the light-sensitive cells of the retina, the photoreceptors. To develop better treatments for patients, we need to understand how inherited retinal disease mutations result in the dysfunction of photoreceptors. New advances in the field of stem cell and gene editing research offer novel ways to model retinal dystrophies in vitro and present opportunities to translate basic biological insights into therapies. This brief review will discuss some of the issues that should be taken into account when carrying out disease modelling and gene editing of retinal cells. We will discuss (i) the use of human induced pluripotent stem cells (iPSCs) for disease modelling and cell therapy; (ii) the importance of using isogenic iPSC lines as controls; (iii) CRISPR/Cas9 gene editing of iPSCs; and (iv) in vivo gene editing using AAV vectors.

Recent findings: Ground-breaking advances in differentiation of iPSCs into retinal organoids and methods to derive mature light sensitive photoreceptors from iPSCs. Furthermore, single AAV systems for in vivo gene editing have been developed which makes retinal in vivo gene editing therapy a real prospect.

Summary: Genome editing is becoming a valuable tool for disease modelling and in vivo gene editing in the retina.

Keywords: Disease modelling; Gene editing; Induced pluripotent stem cells; Photoreceptors; Retina; Vision impairment.

Fig. 1

Schematic diagram depicting how CRISPR/Cas9 gene editing can be harnessed for in vitro disease modelling and in vivo gene editing. CRISPR/Cas9 gene editing panel: Cas9 nuclease in complex with a gRNA can generate specific double-stranded breaks (DSB) in the host DNA. Cell’s DNA repair mechanism will repair the DSB by either error-prone NHEJ or error-free HDR. NHEJ-mediated gene editing will most likely result in the introduction of insertions and deletions (indel, red lines) that will lead to premature STOP codon formation. HDR-mediated gene editing, in the presence of a homologous DNA template (green lines), will introduce precise genomic changes in the host’s DNA. Most popular CRISPR/Cas9 gene editing approaches are an RNP approach, where Cas9 protein is complexed with gRNA for delivery, and a plasmid approach, where Cas9 cDNA, gRNAs and a reporter are usually overexpressed from one, two or more vectors. In vitro disease modelling panel: Shows how fibroblasts can be sampled from healthy and affected individuals and reprogrammed into human iPSCs. CRISPR/Cas9 gene editing can then be used to introduce disease-causing mutations of interest in healthy iPSCs or correct them in affected iPSCs to generate isogenic iPS cell line pairs. These can then be differentiated to photoreceptors for disease modelling purposes. In vivo gene editing: cDNA containing Cas9 gene editing tool can be packed into AAV for subretinal injection to target different populations of retinal cells. The retina is a layered structure composed of three layers of cells connected by two synaptic layers: the inner plexiform layer (IPL) and the outer plexiform layer (OPL). At the outer most region, cone and rod photoreceptor cells form the outer nuclear layer (ONL). The inner nuclear layer (INL) is composed of bipolar, amacrine, horizontal and Müller glia cells. Lastly, the inner most layer, the ganglion cell layer (GCL) is comprised of retinal ganglion cells and displaced amacrine cells. The optic fiber layer (OFL) contains retinal nerve fibers that exit the eye through the optic nerve