https://doi.org/10.1167/tvst.7.5.8 New Developments in Vision Research Translational Retinal Research and Therapies Alison J. Hardcastle1, Paul A. Sieving2, Jose-Alain´ Sahel3, Samuel G. Jacobson4, Artur V. Cideciyan4, John G. Flannery5, William A. Beltran6, and Gustavo D. Aguirre6 1 Professor of Molecular Genetics, UCL Institute of Ophthalmology, London, UK 2 Director, National Eye Institute, National Institutes of Health, Bethesda, MD, USA 3 Department of Ophthalmology, University of Pittsburgh School of Medicine, Director of the UPMC Eye Center, University of Pittsburgh Medical Center, Pittsburgh, PA, USA and Director, Institut de la Vision, Sorbonne Universite-Inserm-CNRS,´ Centre Hospitalier National d’Ophtalmologie des Quinze-Vingts, Paris, France 4 Scheie Eye Institute, Department of Ophthalmology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA 5 Department of Molecular and Cell Biology, Helen Wills Neuroscience Institute, University of California, Berkeley, CA, USA 6 Department of Clinical Sciences and Advanced Medicine, Division of Experimental Retinal Therapies, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, USA Correspondence: Gustavo D. The following review summarizes the state of the art in representative aspects of gene Aguirre, Department of Clinical Sci- therapy/translational medicine and evolves from a symposium held at the School of ences and Advanced Medicine, Di- Veterinary Medicine, University of Pennsylvania on November 16, 2017 honoring Dr. vision of Experimental Retinal Gustavo Aguirre, recipient of ARVO’s 2017 Proctor Medal. Focusing on the retina, Therapies, School of Veterinary speakers highlighted current work on moving therapies for inherited retinal Medicine, University of Pennsylva- degenerative diseases from the laboratory bench to the clinic. nia, Philadelphia, PA 19014, USA. e- mail: [email protected] Received: 23 January 2018 Accepted: 13 April 2018 Published: 13 September 2018 Keywords: retinitis pigmentosa; X- linked Retinoschisis; Leber con- genital amaurosis; animal models Citation: Hardcastle AJ, Sieving PA, Sahel J-A, Jacobson SG, Cideciyan AV, Flannery JG, Beltran WA, Aguirre GD. Translational retinal research and therapies. Trans Vis Sci Tech. 2018;7(5):8, https://doi.org/10.1167/ tvst.7.5.8 Copyright 2018 The Authors Defining the Inherited Retinal ical or benign. Nevertheless, next-generation sequenc- ing offers the opportunity to reveal the unusual and Dystrophy Genome unexpected, such as expansion of phenotypes associ- ated with specific genes, unexpected inheritance Professor Alison J. Hardcastle patterns and aberrant splicing, which is emerging as Abstract: Inherited retinal dystrophies display re- a relatively common mechanism of disease resulting markable genetic heterogeneity, reflecting the com- from introduction of cryptic exons, and rare combi- plexity of retinal structure and function. Next- nations of single-nucleotide polymorphism haplo- generation sequencing technologies have transformed types. As a complementary approach to developing our ability to identify mutation-causing diseases but animal models to study disease mechanisms and test also present additional challenges of interpreting potential mutation-specific therapies, we can differ- exome and genome variants as potentially patholog- entiate pluripotent stem cells to retinal pigment 1 TVST j 2018 j Vol. 7 j No. 5 j Article 8 This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License. Hardcastle et al. Figure 1. Mapped and identified retinal disease genes. Figure modified from RetNet (in the public domain, https://sph.uth.edu/retnet) to highlight landmark time points in path to disease gene discovery. epithelium and optic cup organoids with mutations in of genes (~20,000 genes), can identify genetic causes human genomic and cellular context, another very in approximately 60% of cases. However, all 3 billion useful tool in the study of inherited retinal degener- nucleotides can now be sequenced using whole- ations. genome sequencing (WGS), but it can be very challenging to identify a causative mutation as WGS The first gene mutation causing an inherited retinal uncovers approximately 5 million differences in an degeneration was reported in the rhodopsin gene in individual compared with the reference human 1990.1 Linkage studies and Sanger sequencing of genome. Nevertheless, NGS has provided an unbiased candidate genes were instrumental in these early opportunity to reveal unusual and unexpected phe- analyses, but the completion of the human genome notypic associations, inheritance patterns, and genetic sequence and next-generation sequencing (NGS) mechanisms of disease (see examples below). Finally, technologies has accelerated the rate of discovery. animal models of retinal degeneration, particularly in Today, over 280 retinal disease genes have been the dog, have been instrumental in studying orthol- mapped and/or identified (Fig. 1). These retinal gene ogous human retinal degenerations. For example, in mutations lead to many different types of retinal XLPRA,2 physical mapping of the canine genome, dystrophy, including Leber congenital amaurosis linkage mapping and cloning with characterization of (LCA), which affects rod and cone photoreceptors, the RPGR gene, led to the identification of the cone-specific diseases, such as achromatopsia, macu- causative RPGR variants in different dog breeds and lar degeneration, such as Stargardt disease, and rare finally to gene therapy.3 Several canine disease models syndromic diseases, such as Usher Syndrome and have been developed by the Aguirre group, including Bardet-Biedl Syndrome. Mutations in a diverse models of photoreceptor dysplasia (1998), RPE65- number of genes can lead to a similar disease based childhood blindness (2001), and achromatopsia phenotype; for example, at least 14 genes have been (2002); stationary night blindness has even been identified whose phenotypic characteristics fall within studied in the Appaloosa horse (1978). the LCA classification. NGS has accelerated this gene As mentioned above, NGS is a very valuable tool discovery process, but this also presents challenges in in studying retinal degenerations because it is interpreting potentially pathogenic variants compared unbiased and allows for all possible inheritance with benign variants. Specifically, whole-exome se- modes to be considered for association of gene quencing, which will sequence all protein coding parts variants with novel phenotypes. A good example of 2 TVST j 2018 j Vol. 7 j No. 5 j Article 8 Hardcastle et al. this is the study of missense variants in the X-linked 3.7 All in all, it is clear that mutations in the LW and gene PRPS1, which can cause retinal degeneration in MW cone genes can lead to a wide variety of females.4 These patients show a type of retinitis dystrophies ranging from simple color blindness to pigmentosa (RP) with tapetal-like reflexes and severe cone cell dystrophies. patchy retinal atrophy. Missense mutations in Along with mutant gene discovery and character- PRPS1 can have multiple effects, causing Arts ization, drugs and small molecules targeting specific Syndrome, X-linked Charcot-Marie Tooth 5, and gene mutations, such as translational read-through X-linked nonsyndromic deafness. The absence of (TR-T) drugs, can be used in therapy. An example is affectedmalesintheRPfamiliessuggestssome the case of mutations in the RP2 gene, which leads to PRPS1 variants may be embryonic male lethal when vision loss in a form of X-linked RP, and is often inherited in the hemizygous state. In another family affected by nonsense mutations. Patient fibroblasts consistent with X-linked inheritance, no causative and iPSC-derived retinal pigment epithelial (RPE) variants on the X-chromosome were found; however, cells from a patient with the R120X mutation lack two potentially deleterious compound heterozygous RP2 protein, but, using translational read-through- variants were detected in two autosomal recessive inducing drugs, up to 20% of normal RP2 protein can genes, indicating two recessive conditions in different be restored8 with reversal of cellular phenotype branches of the family. Similarly, linkage analyses defects. This suggests that the TR-T drugs could be and NGS can uncover new loci and genes for tested clinically for the restoration of vision in some genetically heterogeneous diseases. For example, RP2 patients with nonsense mutations. In a similar the identification of the mutation responsible for manner, specific gene mutations can be studied in the RP23 locus for X-linked RP5 was possible patient iPSC-derived RPE and optic cups in genomic through targeted genomic NGS that revealed a deep and cellular context, providing a model system to test intronic mutation in OFD1.6 Point mutations in the disease mechanisms and potential therapies. In optic coding region and splice sites of OFD1 cause cups, not only can the underlying defects be ciliopathies, including male-lethal orofaciodigital identified, but corrective measures can also be syndrome 1, Simpson-Golabi-Behmel syndrome, designed and tested. Three-dimensional optic cups type 2, and Joubert syndrome. Interestingly, RP23 from iPSCs carrying a CEP290 mutation that causes is not male lethal; lower levels of transcript are LCA revealed that the optic cups have higher levels of present, perhaps producing enough normal protein aberrant splicing caused by the intronic mutation for other ciliated