Alexander J. Smith

The Rpe65-deficient dog has been important for development of translational therapies of Leber congenital amaurosis type 2 (LCA2). The purpose of this study was to provide a comprehensive report of the natural history of retinal changes in this dog model. Rpe65-deficient dogs from 2 months to 10 years of age were assessed by fundus imaging, electroretinography (ERG) and vision testing (VT). Changes in retinal layer thickness were assessed by optical coherence tomography and on plastic retinal sections. ERG showed marked loss of retinal sensitivity, with amplitudes declining with age. Retinal thinning initially developed in the area centralis, with a slower thinning of the outer retina in other areas starting with the inferior retina. VT showed that dogs of all ages performed well in bright light, while at lower light levels they were blind. Retinal pigment epithelial (RPE) inclusions developed and in younger dogs and increased in size with age. The loss of photoreceptors was mirrored by a decline in ERG amplitudes. The slow degeneration meant that sufficient photoreceptors, albeit very desensitized, remained to allow for residual bright light vision in older dogs. This study shows the natural history of the Rpe65-deficient dog model of LCA2.

Mutations in retinitis pigmentosa GTPase regulator (RPGR) cause severe retinal ciliopathy, X-linked retinitis pigmentosa. Although two major alternatively spliced isoforms, RPGR^ex1-19 and RPGR^ORF15, are expressed, the relative importance of these isoforms in disease pathogenesis is unclear. Here, we analyzed fibroblast samples from eight patients and found that all of them form longer cilia than normal controls, albeit to different degrees. Although all mutant RPGR^ORF15 messenger RNAs (mRNAs) are unstable, their steady-state levels were similar or higher than those in the control cells, suggesting there may be increased transcription. Three of the fibroblasts that had higher levels of mutant RPGR^ORF15 mRNA also exhibited significantly higher levels of RPGR^ex1-19 mRNA. Four samples with unaltered RPGR^ex1-19 levels carried mutations in RPGR^ORF15 that resulted in this isoform being relatively less stable. Thus, in all cases, the RPGR^ex1-19/RPGR^ORF15 isoform ratio was increased, and this was highly correlative to the cilia extension defect. Moreover, overexpression of RPGR^ex1-19 (mimicking the increase in RPGR^ex1-19 to RPGR^ORF15 isoform ratio) or RPGR^ORF15 (mimicking reduction of the ratio) resulted in significantly longer or shorter cilia, respectively. Notably, the cilia length defect appears to be attributable to both the loss of the wild-type RPGR^ORF15 protein and to the higher levels of the RPGR^ex1-19 isoform, indicating that the observed defect is due to the altered isoform ratios. These results suggest that maintaining the optimal RPGR^ex1-9 to RPGR^ORF15 ratio is critical for cilia growth and that designing strategies that focus on the best ways to restore the RPGR^ex1-19/RPGR^ORF15 ratio may lead to better therapeutic outcomes.

The neuronal ceroid lipofuscinoses (NCLs), often referred to as Batten disease, are inherited lysosomal storage disorders that represent the most common neurodegeneration during childhood. Symptoms include seizures, vision loss, motor and cognitive decline and premature death. The development of brain-directed treatments for NCLs has made noteworthy progress in recent years. Clinical trials are currently ongoing or planned for different forms of the disease. Despite these promising advances, it is unlikely that therapeutic interventions targeting the brain will prevent loss of vision in patients as retinal cells remain untreated and will continue to degenerate. Here, we demonstrate that Cln3Δex7/8 mice, a mouse model of CLN3 Batten disease with juvenile onset, suffer from a decline in inner retinal function resulting from the death of rod bipolar cells, interneurons vital for signal transmission from photoreceptors to ganglion cells in the retina. We also show that this ocular phenotype can be treated by adeno-associated virus (AAV)-mediated expression of CLN3 in cells of the inner retina, leading to significant survival of bipolar cells and preserved retinal function. In contrast, the treatment of photoreceptors, which are lost in patients at late disease stages, was not therapeutic in Cln3Δex7/8 mice, underlining the notion that CLN3 disease is primarily a disease of the inner retina with secondary changes in the outer retina. These data indicate that bipolar cells play a central role in this disease and identify this cell type as an important target for ocular AAV-based gene therapies for CLN3 disease.

Rhodopsin misfolding caused by the P23H mutation is a major cause of autosomal dominant retinitis pigmentosa (adRP). To date, there are no effective treatments for adRP. The BiP co-chaperone and reductase ERdj5 (DNAJC10) is part of the endoplasmic reticulum (ER) quality control machinery, and previous studies have shown that overexpression of ERdj5 in vitro enhanced the degradation of P23H rhodopsin, whereas knockdown of ERdj5 increased P23H rhodopsin ER retention and aggregation. Here, we investigated the role of ERdj5 in photoreceptor homeostasis in vivo by using an Erdj5 knockout mouse crossed with the P23H knock-in mouse and by adeno-associated viral (AAV) vector-mediated gene augmentation of ERdj5 in P23H-3 rats. Electroretinogram (ERG) and optical coherence tomography of Erdj5^−/− and P23H^+/−:Erdj5^−/− mice showed no effect of ERdj5 ablation on retinal function or photoreceptor survival. Rhodopsin levels and localization were similar to those of control animals at a range of time points. By contrast, when AAV2/8-ERdj5-HA was subretinally injected into P23H-3 rats, analysis of the full-field ERG suggested that overexpression of ERdj5 reduced visual function loss 10 weeks post-injection (PI). This correlated with a significant preservation of photoreceptor cells at 4 and 10 weeks PI. Assessment of the outer nuclear layer (ONL) morphology showed preserved ONL thickness and reduced rhodopsin retention in the ONL in the injected superior retina. Overall, these data suggest that manipulation of the ER quality control and ER-associated degradation factors to promote mutant protein degradation could be beneficial for the treatment of adRP caused by mutant rhodopsin.

Cone photoreceptors mediate daylight vision and are the primary cells responsible for vision in humans. Cone dysfunction leads to poor quality daylight vision because rod photoreceptors become saturated and non-functional at high light levels. Here we demonstrate that in mice lacking cone function, AAV-mediated over-expression of Rgs9-anchor protein (R9AP), a critical component of the GTPase complex that mediates the deactivation of the phototransduction cascade, results in desensitization of rod function and a “photopic shift” of the rod-driven electroretinogram. This treatment enables rods to respond to brighter light (up to ∼2.0 log) with increased visually-evoked cortical responses to high intensity stimulation. These results suggest that AAV-mediated transfer of R9ap into rods might be used to improve daylight vision in humans visually handicapped by cone dysfunction.

The use of human pluripotent stem cell-derived retinal cells for cell therapy strategies and disease modelling relies on the ability to obtain healthy and organised retinal tissue in sufficient quantities. Generating such tissue is a lengthy process, often taking over 6 months of cell culture, and current approaches do not always generate large quantities of the major retinal cell types required. We adapted our previously described differentiation protocol to investigate the use of stirred-tank bioreactors. We used immunohistochemistry, flow cytometry and electron microscopy to characterise retinal organoids grown in standard and bioreactor culture conditions. Our analysis revealed that the use of bioreactors results in improved laminar stratification as well as an increase in the yield of photoreceptor cells bearing cilia and nascent outer-segment-like structures. Bioreactors represent a promising platform for scaling up the manufacture of retinal cells for use in disease modelling, drug screening and cell transplantation studies.

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.

Over the last decade, gene supplementation therapy for inherited retinal degeneration has come of age. Early proof-of-concept studies in animal models of disease showed modest, but genuine improvements in retinal function and/or survival. Further development of the vectors used for gene transfer to the retina has led to better treatment efficacy in a wide variety of animal models, leading in 2008 to the initiation of three clinical trials for Leber congenital amaurosis caused by retinal pigment epithelium 65 deficiency. The results from these trials suggest that the treatment of inherited retinal dystrophy by gene therapy can be safe and effective. Here, we examine the progress of gene supplementation therapy in the retina, and discuss the potential for using gene therapy to treat different forms of inherited retinal degeneration.

To date adeno-associated viral (AAV) vectors are the only gene therapy vectors that have been shown to efficiently transduce photoreceptor cells and have thus become the most commonly used vector for ocular transduction. Various AAV serotypes have been evaluated in the eye, the first of which was AAV2, which is able to transduce photoreceptors, retinal pigment epithelium (RPE) and retinal ganglion cells. AAV serotypes 1 and 4, as well as AAV2 pseudotyped with these capsids, only transduce the RPE. AAV serotype 5 and AAV2/5 transduce the photoreceptors as well as RPE, but not retinal ganglion cells. Here, we assessed the capacity of the novel serotype AAV2/8 to transduce various ocular tissues of the adult murine retina by administering AAV2/8 green fluorescent protein intravitreally, subretinally and intracamerally. We also determined the kinetics and efficiency of self-complementary AAV (scAAV) vectors of serotypes 2/2, 2/5 and 2/8 and compared them with single-stranded AAV (ssAAV). We found that ssAAV2/8 transduces photoreceptors and RPE more efficiently than ssAAV2/2 and ssAAV2/5, and that scAAV2/8 had faster onset and higher transgene expression than ssAAV2/8. This improved transduction efficiency might facilitate the development of improved gene therapy protocols for inherited retinal degenerations, particularly those caused by defects in photoreceptor-specific genes.