Inherited Retinal Degeneration Caused by Dehydrodolichyl Diphosphate Synthase Mutation-Effect of an ALG6 Modifier Variant.

Modern advances in disease genetics have uncovered numerous modifier genes that play a role in the severity of disease expression. One such class of genetic conditions is known as inherited retinal degenerations (IRDs), a collection of retinal degenerative disorders caused by mutations in over 300 genes. A single missense mutation (K42E) in the gene encoding the enzyme dehydrodolichyl diphosphate synthase (DHDDS), which is required for protein N-glycosylation in all cells and tissues, causes DHDDS-IRD (retinitis pigmentosa type 59 (RP59; OMIM #613861)). Apart from a retinal phenotype, however, DHDDS-IRD is surprisingly non-syndromic (i.e., without any systemic manifestations). To explore disease pathology, we selected five glycosylation-related genes for analysis that are suggested to have disease modifier variants. These genes encode glycosyltransferases (ALG6, ALG8), an ER resident protein (DDOST), a high-mannose oligosaccharyl transferase (MPDU1), and a protein N-glycosylation regulatory protein (TNKS). DNA samples from 11 confirmed DHDDS (K42E)-IRD patients were sequenced at the site of each candidate genetic modifier. Quantitative measures of retinal structure and function were performed across five decades of life by evaluating foveal photoreceptor thickness, visual acuity, foveal sensitivity, macular and extramacular rod sensitivity, and kinetic visual field extent. The ALG6 variant, (F304S), was correlated with greater macular cone disease severity and less peripheral rod disease severity. Thus, modifier gene polymorphisms may account for a significant portion of phenotypic variation observed in human genetic disease. However, the consequences of the polymorphisms may be counterintuitively complex in terms of rod and cone populations affected in different regions of the retina.

Gene therapy reforms photoreceptor structure and restores vision in NPHP5-associated Leber congenital amaurosis.

The inherited childhood blindness caused by mutations in NPHP5, a form of Leber congenital amaurosis, results in abnormal development, dysfunction, and degeneration of photoreceptors. A naturally occurring NPHP5 mutation in dogs leads to a phenotype that very nearly duplicates the human retinopathy in terms of the photoreceptors involved, spatial distribution of degeneration, and the natural history of vision loss. We show that adeno-associated virus (AAV)-mediated NPHP5 gene augmentation of mutant canine retinas at the time of active degeneration and peak cell death stably restores photoreceptor structure, function, and vision with either the canine or human NPHP5 transgenes. Mutant cone photoreceptors, which failed to form outer segments during development, reform this structure after treatment. Degenerating rod photoreceptor outer segments are stabilized and develop normal structure. This process begins within 8 weeks after treatment and remains stable throughout the 6-month posttreatment period. In both photoreceptor cell classes mislocalization of rod and cone opsins is minimized or reversed. Retinal function and functional vision are restored. Efficacy of gene therapy in this large animal ciliopathy model of Leber congenital amaurosis provides a path for translation to human treatment.

Long-Term Structural Outcomes of Late-Stage RPE65 Gene Therapy.

The form of hereditary childhood blindness Leber congenital amaurosis (LCA) caused by biallelic RPE65 mutations is considered treatable with a gene therapy product approved in the US and Europe. The resulting vision improvement is well accepted, but long-term outcomes on the natural history of retinal degeneration are controversial. We treated four RPE65-mutant dogs in mid-life (age = 5-6 years) and followed them long-term (4-5 years). At the time of the intervention at mid-life, there were intra-ocular and inter-animal differences in local photoreceptor layer health ranging from near normal to complete degeneration. Treated locations having more than 63% of normal photoreceptors showed robust treatment-related retention of photoreceptors in the long term. Treated regions with less retained photoreceptors at the time of the intervention showed progressive degeneration similar to untreated regions with matched initial stage of disease. Unexpectedly, both treated and untreated regions in study eyes tended to show less degeneration compared to matched locations in untreated control eyes. These results support the hypothesis that successful long-term arrest of progression with RPE65 gene therapy may only occur in retinal regions with relatively retained photoreceptors at the time of the intervention, and there may be heretofore unknown mechanisms causing long-distance partial treatment effects beyond the region of subretinal injection.

BEST1 gene therapy corrects a diffuse retina-wide microdetachment modulated by light exposure.

Mutations in the BEST1 gene cause detachment of the retina and degeneration of photoreceptor (PR) cells due to a primary channelopathy in the neighboring retinal pigment epithelium (RPE) cells. The pathophysiology of the interaction between RPE and PR cells preceding the formation of retinal detachment remains not well-understood. Our studies of molecular pathology in the canine BEST1 disease model revealed retina-wide abnormalities at the RPE-PR interface associated with defects in the RPE microvillar ensheathment and a cone PR-associated insoluble interphotoreceptor matrix. In vivo imaging demonstrated a retina-wide RPE-PR microdetachment, which contracted with dark adaptation and expanded upon exposure to a moderate intensity of light. Subretinal BEST1 gene augmentation therapy using adeno-associated virus 2 reversed not only clinically detectable subretinal lesions but also the diffuse microdetachments. Immunohistochemical analyses showed correction of the structural alterations at the RPE-PR interface in areas with BEST1 transgene expression. Successful treatment effects were demonstrated in three different canine BEST1 genotypes with vector titers in the 0.1-to-5E11 vector genomes per mL range. Patients with biallelic BEST1 mutations exhibited large regions of retinal lamination defects, severe PR sensitivity loss, and slowing of the retinoid cycle. Human translation of canine BEST1 gene therapy success in reversal of macro- and microdetachments through restoration of cytoarchitecture at the RPE-PR interface has promise to result in improved visual function and prevent disease progression in patients affected with bestrophinopathies.

Mutation-independent rhodopsin gene therapy by knockdown and replacement with a single AAV vector.

Inherited retinal degenerations are caused by mutations in >250 genes that affect photoreceptor cells or the retinal pigment epithelium and result in vision loss. For autosomal recessive and X-linked retinal degenerations, significant progress has been achieved in the field of gene therapy as evidenced by the growing number of clinical trials and the recent commercialization of the first gene therapy for a form of congenital blindness. However, despite significant efforts to develop a treatment for the most common form of autosomal dominant retinitis pigmentosa (adRP) caused by >150 mutations in the rhodopsin (RHO) gene, translation to the clinic has stalled. Here, we identified a highly efficient shRNA that targets human (and canine) RHO in a mutation-independent manner. In a single adeno-associated viral (AAV) vector we combined this shRNA with a human RHO replacement cDNA made resistant to RNA interference and tested this construct in a naturally occurring canine model of RHO-adRP. Subretinal vector injections led to nearly complete suppression of endogenous canine RHO RNA, while the human RHO replacement cDNA resulted in up to 30% of normal RHO protein levels. Noninvasive retinal imaging showed photoreceptors in treated areas were completely protected from retinal degeneration. Histopathology confirmed retention of normal photoreceptor structure and RHO expression in rod outer segments. Long-term (>8 mo) follow-up by retinal imaging and electroretinography indicated stable structural and functional preservation. The efficacy of this gene therapy in a clinically relevant large-animal model paves the way for treating patients with RHO-adRP.

Leber Congenital Amaurosis Due to GUCY2D Mutations: Longitudinal Analysis of Retinal Structure and Visual Function.

Gene augmentation therapy is being planned for GUCY2D-associated Leber congenital amaurosis (LCA). To increase our understanding of the natural history of GUCY2D-LCA, patients were evaluated twice with an interval of 4 to 7 years between visits using safety and efficacy outcome measures previously determined to be useful for monitoring this disorder. In this group of molecularly-identified LCA patients (n = 10; ages 7-37 years at first visit), optical coherence tomography (OCT) was used to measure foveal cone outer nuclear layer (ONL) thickness and rod ONL at a superior retinal locus. Full-field stimulus testing (FST) with chromatic stimuli in dark- and light-adapted states was used to assay rod and cone vision. Changes in OCT and FST over the interval were mostly attributable to inter-visit variability. There were no major negative changes in structure or function across the cohort and over the intervals studied. Variation in severity of disease expression between patients occurs; however, despite difficulties in quantifying structure and function in such seriously visually impaired individuals with nystagmus, the present work supports the use of OCT as a safety outcome and FST as an efficacy outcome in a clinical trial of GUCY2D-LCA. A wide age spectrum for therapy was confirmed, and there was relative stability of structure and function during a typical time interval for clinical trials.

Autosomal Dominant Retinitis Pigmentosa Due to Class B Rhodopsin Mutations: An Objective Outcome for Future Treatment Trials.

Gene therapy for adRP due to RHO mutations was recently shown to prevent photoreceptor death in a canine model of Class B disease. Among translational steps to be taken, one is to determine a method to detect efficacy in a human clinical trial. The relatively slow progression of adRP becomes a difficulty for clinical trials requiring an answer to whether there is slowed progression of degeneration in response to therapy. We performed a single-center, retrospective observational study of cross-sectional and longitudinal data. The study was prompted by our identification of a pericentral disease distribution in Class B RHO-adRP. Ultrawide optical coherence tomography (OCT) scans were used. Inferior retinal pericentral defects was an early disease feature. Degeneration further inferior in the retina merged with the pericentral defect, which extended into superior retina. In about 70% of patients, there was an asymmetric island of structure with significantly greater superior than inferior ellipsoid zone (EZ) extent. Serial measures of photoreceptor structure by OCT indicated constriction in superior retinal extent within a two-year interval. We conclude that these results should allow early-phase trials of therapy in RHO-adRP to move forward by inclusion of patients with an asymmetric extent of photoreceptor structure and by monitoring therapeutic effects over two years in the superior retina, a reasonable target for subretinal injection.

Variegated yet non-random rod and cone photoreceptor disease patterns in RPGR-ORF15-associated retinal degeneration.

Mutations in the ORF15 exon of the RPGR gene cause a common form of X-linked retinitis pigmentosa, which often results in severe loss of vision. In dogs and mice, gene augmentation therapy has been shown to arrest the progressive degeneration of rod and cone photoreceptors. However, the distribution of potentially treatable photoreceptors across the human retinas and the rate of degeneration are not known. Here, we have defined structural and functional features of the disease in 70 individuals with ORF15 mutations. We also correlated the features observed in patients with those of three Rpgr-mutant (Rpgr-ko, Rd9, and Rpgr-cko) mice. In patients, there was pronounced macular disease. Across the retina, rod and cone dysfunction showed a range of patterns and a spectrum of severity between individuals, but a high symmetry was observed between eyes of each individual. Genotype was not related to disease expression. In the Rpgr-ko mice, there were intra-retinal differences in rhodopsin and cone opsin trafficking. In Rd9 and Rpgr-cko mice, retinal degeneration showed inter-ocular symmetry. Longitudinal results in patients revealed localized rod and cone dysfunction with progression rates of 0.8 to 1.3 log per decade in sensitivity loss. Relatively retained rod and cone photoreceptors in mid- and far-peripheral temporal-inferior and nasal-inferior visual field regions should be good targets for future localized gene therapies in patients.

Overlap of abnormal photoreceptor development and progressive degeneration in Leber congenital amaurosis caused by NPHP5 mutation.

Ciliary defects can result in severe disorders called ciliopathies. Mutations in NPHP5 cause a ciliopathy characterized by severe childhood onset retinal blindness, Leber congenital amaurosis (LCA), and renal disease. Using the canine NPHP5-LCA model we compared human and canine retinal phenotypes, and examined the early stages of photoreceptor development and degeneration, the kinetics of photoreceptor loss, the progression of degeneration and the expression profiles of selected genes. NPHP5-mutant dogs recapitulate the human phenotype of very early loss of rods, and relative retention of the central retinal cone photoreceptors that lack function. In mutant dogs, rod and cone photoreceptors have a sensory cilium, but develop and function abnormally and then rapidly degenerate; L/M cones are more severely affected than S-cones. The lack of outer segments in mutant cones indicates a ciliary dysfunction. Genes expressed in mutant rod or both rod and cone photoreceptors show significant downregulation, while those expressed only in cones are unchanged. Many genes in cell-death and -survival pathways also are downregulated. The canine disease is a non-syndromic LCA-ciliopathy, with normal renal structures and no CNS abnormalities. Our results identify the critical time points in the pathogenesis of the photoreceptor disease, and bring us closer to defining a potential time window for testing novel therapies for translation to patients.

Optimization of Retinal Gene Therapy for X-Linked Retinitis Pigmentosa Due to RPGR Mutations.

X-linked retinitis pigmentosa (XLRP) caused by mutations in the RPGR gene is an early onset and severe cause of blindness. Successful proof-of-concept studies in a canine model have recently shown that development of a corrective gene therapy for RPGR-XLRP may now be an attainable goal. In preparation for a future clinical trial, we have here optimized the therapeutic AAV vector construct by showing that GRK1 (rather than IRBP) is a more efficient promoter for targeting gene expression to both rods and cones in non-human primates. Two transgenes were used in RPGR mutant (XLPRA2) dogs under the control of the GRK1 promoter. First was the previously developed stabilized human RPGR (hRPGRstb). Second was a new full-length stabilized and codon-optimized human RPGR (hRPGRco). Long-term (>2 years) studies with an AAV2/5 vector carrying hRPGRstb under control of the GRK1 promoter showed rescue of rods and cones from degeneration and retention of vision. Shorter term (3 months) studies demonstrated comparable preservation of photoreceptors in canine eyes treated with an AAV2/5 vector carrying either transgene under the control of the GRK1 promoter. These results provide the critical molecular components (GRK1 promoter, hRPGRco transgene) to now construct a therapeutic viral vector optimized for RPGR-XLRP patients.

Successful arrest of photoreceptor and vision loss expands the therapeutic window of retinal gene therapy to later stages of disease.

Inherited retinal degenerations cause progressive loss of photoreceptor neurons with eventual blindness. Corrective or neuroprotective gene therapies under development could be delivered at a predegeneration stage to prevent the onset of disease, as well as at intermediate-degeneration stages to slow the rate of progression. Most preclinical gene therapy successes to date have been as predegeneration interventions. In many animal models, as well as in human studies, to date, retinal gene therapy administered well after the onset of degeneration was not able to modify the rate of progression even when successfully reversing dysfunction. We evaluated consequences of gene therapy delivered at intermediate stages of disease in a canine model of X-linked retinitis pigmentosa (XLRP) caused by a mutation in the Retinitis Pigmentosa GTPase Regulator (RPGR) gene. Spatiotemporal natural history of disease was defined and therapeutic dose selected based on predegeneration results. Then interventions were timed at earlier and later phases of intermediate-stage disease, and photoreceptor degeneration monitored with noninvasive imaging, electrophysiological function, and visual behavior for more than 2 y. All parameters showed substantial and significant arrest of the progressive time course of disease with treatment, which resulted in long-term improved retinal function and visual behavior compared with control eyes. Histology confirmed that the human RPGR transgene was stably expressed in photoreceptors and associated with improved structural preservation of rods, cones, and ON bipolar cells together with correction of opsin mislocalization. These findings in a clinically relevant large animal model demonstrate the long-term efficacy of RPGR gene augmentation and substantially broaden the therapeutic window for intervention in patients with RPGR-XLRP.

Protein misfolding and the pathogenesis of ABCA4-associated retinal degenerations.

Mutations in the ABCA4 gene are a common cause of autosomal recessive retinal degeneration. All mouse models to date are based on knockouts of Abca4, even though the disease is often caused by missense mutations such as the complex allele L541P;A1038V (PV). We now show that the PV mutation causes severe human disease whereas the V mutation alone causes mild disease. Mutant ABCA4 proteins expressed heterologously in mammalian cells retained normal cellular localization. However, basal and all-trans-retinal-stimulated ATPase activities were reduced substantially for P and PV but only mildly for V. Electron microscopy revealed marked structural changes and misfolding for the P and PV mutants but few changes for the V mutant, consistent with the disease severity difference in patients. We generated Abca4(PV/PV) knock-in mice homozygous for the complex PV allele to investigate the effects of this misfolding mutation in vivo. Mutant ABCA4 RNA levels approximated WT ABCA4 RNA levels but, surprisingly, only trace amounts of mutant ABCA4 protein were noted in the retina. RNA sequencing of WT, Abca4(-/-) and Abca4(PV/PV) mice revealed mild gene expression alterations in the retina and RPE. Similar to Abca4(-/-) mice, Abca4(PV/PV) mice showed substantial A2E and lipofuscin accumulation in their RPE cells but no retinal degeneration up to 12 months of age. Thus, rapid degradation of this large misfolded mutant protein in mouse retina caused little detectable photoreceptor degeneration. These findings suggest likely differences in the unfolded protein response between murine and human photoreceptors and support development of therapies directed at increasing this capability in patients.

Human retinal gene therapy for Leber congenital amaurosis shows advancing retinal degeneration despite enduring visual improvement.

Leber congenital amaurosis (LCA) associated with retinal pigment epithelium-specific protein 65 kDa (RPE65) mutations is a severe hereditary blindness resulting from both dysfunction and degeneration of photoreceptors. Clinical trials with gene augmentation therapy have shown partial reversal of the dysfunction, but the effects on the degeneration are not known. We evaluated the consequences of gene therapy on retinal degeneration in patients with RPE65-LCA and its canine model. In untreated RPE65-LCA patients, there was dysfunction and degeneration of photoreceptors, even at the earliest ages. Examined serially over years, the outer photoreceptor nuclear layer showed progressive thinning. Treated RPE65-LCA showed substantial visual improvement in the short term and no detectable decline from this new level over the long term. However, retinal degeneration continued to progress unabated. In RPE65-mutant dogs, the first one-quarter of their lifespan showed only dysfunction, and there was normal outer photoreceptor nuclear layer thickness retina-wide. Dogs treated during the earlier dysfunction-only stage showed improved visual function and dramatic protection of treated photoreceptors from degeneration when measured 5-11 y later. Dogs treated later during the combined dysfunction and degeneration stage also showed visual function improvement, but photoreceptor loss continued unabated, the same as in human RPE65-LCA. The results suggest that, in RPE65 disease treatment, protection from visual function deterioration cannot be assumed to imply protection from degeneration. The effects of gene augmentation therapy are complex and suggest a need for a combinatorial strategy in RPE65-LCA to not only improve function in the short term but also slow retinal degeneration in the long term.

Gene therapy rescues photoreceptor blindness in dogs and paves the way for treating human X-linked retinitis pigmentosa.

Hereditary retinal blindness is caused by mutations in genes expressed in photoreceptors or retinal pigment epithelium. Gene therapy in mouse and dog models of a primary retinal pigment epithelium disease has already been translated to human clinical trials with encouraging results. Treatment for common primary photoreceptor blindness, however, has not yet moved from proof of concept to the clinic. We evaluated gene augmentation therapy in two blinding canine photoreceptor diseases that model the common X-linked form of retinitis pigmentosa caused by mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene, which encodes a photoreceptor ciliary protein, and provide evidence that the therapy is effective. After subretinal injections of adeno-associated virus-2/5-vectored human RPGR with human IRBP or GRK1 promoters, in vivo imaging showed preserved photoreceptor nuclei and inner/outer segments that were limited to treated areas. Both rod and cone photoreceptor function were greater in treated (three of four) than in control eyes. Histopathology indicated normal photoreceptor structure and reversal of opsin mislocalization in treated areas expressing human RPGR protein in rods and cones. Postreceptoral remodeling was also corrected: there was reversal of bipolar cell dendrite retraction evident with bipolar cell markers and preservation of outer plexiform layer thickness. Efficacy of gene therapy in these large animal models of X-linked retinitis pigmentosa provides a path for translation to human treatment.

Retinal response to light exposure in BEST1-mutant dogs evaluated with ultra-high resolution OCT.

Mutations in BEST1 cause an autosomal recessive disease in dogs where the earliest changes localize to the photoreceptor-RPE interface and show a retina-wide micro-detachment that is modulated by light exposure. The purpose of this study was to define the spatial and temporal details of the outer retina and its response to light with ultra-high resolution OCT across a range of ages and with different BEST1 mutations. Three retinal regions were selected in each eye: near the fovea-like area, near the optic nerve, both in the tapetal area, and inferior to the optic nerve in the non-tapetal area. The OS+ slab thickness was defined between the peak near the junction of inner and outer segments (IS/OS) and the transition between basal RPE, Bruch membrane, choriocapillaris and proximal tapetum (RPE/T). In wildtype (WT) dogs, two tapetal regions showed additional hyperscattering OCT peaks within the OS+ slab likely representing cone and rod outer segment tips (COST and ROST). The inferior non-tapetal region of WT dogs had only one of these peaks, likely ROST. In dogs with BEST1 mutations, all three locations showed a single peak, likely suggesting optical silence of COST. Light-dependent expansion of the micro-detachment by about 10 um was detectable in both tapetal and non-tapetal retina across all ages and BEST1 mutations.

Cone photoreceptors are the main targets for gene therapy of NPHP5 (IQCB1) or NPHP6 (CEP290) blindness: generation of an all-cone Nphp6 hypomorph mouse that mimics the human retinal ciliopathy.

Leber congenital amaurosis (LCA), a severe autosomal recessive childhood blindness, is caused by mutations in at least 15 genes. The most common molecular form is a ciliopathy due to NPHP6 (CEP290) mutations and subjects have profound loss of vision. A similarly severe phenotype occurs in the related ciliopathy NPHP5 (IQCB1)-LCA. Recent success of retinal gene therapy in one form of LCA prompted the question whether we know enough about human NPHP5 and NPHP6 disease to plan such treatment. We determined that there was early-onset rapid degeneration of rod photoreceptors in young subjects with these ciliopathies. Rod outer segment (OS) lamination, when detectable, was disorganized. Retinal pigment epithelium lipofuscin accumulation indicated that rods had existed in the past in most subjects. In contrast to early rod losses, the all-cone human fovea in NPHP5- and NPHP6-LCA of all ages retained cone nuclei, albeit with abnormal inner segments and OS. The rd16 mouse, carrying a hypomorphic Nphp6 allele, was a good model of the rod-dominant human extra-foveal retina. Rd16 mice showed normal genesis of photoreceptors, including the formation of cilia, followed by abnormal elaboration of OS and rapid degeneration. To produce a model of the all-cone human fovea in NPHP6-LCA, we generated rd16;Nrl-/- double-mutant mice. They showed substantially retained cone photoreceptors with disproportionate cone function loss, such as in the human disease. NPHP5- and NPHP6-LCA across a wide age spectrum are thus excellent candidates for cone-directed gene augmentation therapy, and the rd16;Nrl-/- mouse is an appropriate model for pre-clinical proof-of-concept studies.

Durable vision improvement after a single intravitreal treatment with antisense oligonucleotide in CEP290-LCA: Replication in two eyes.

Purpose: An intravitreally injected antisense oligonucleotide, sepofarsen, was designed to modulate splicing within retinas of patients with severe vision loss due to deep intronic c.2991 + 1655A > G variant in the CEP290 gene. A previous report showed vision improvements following a single injection in one eye with unexpected durability lasting at least 15 months. The current study evaluated durability of efficacy beyond 15 months in the previously treated left eye. In addition, peak efficacy and durability were evaluated in the treatment-naive right eye, and re-injection of the left eye 4 years after the first injection. Observations: Visual function was evaluated with best corrected standard and low-luminance visual acuities, microperimetry, dark-adapted chromatic perimetry, and full-field sensitivity testing. Retinal structure was evaluated with OCT imaging. At the fovea, all visual function measures and IS/OS intensity of the OCT showed transient improvements peaking at 3-6 months, remaining better than baseline at ∼2 years, and returning to baseline by 3-4 years after each single injection. Conclusions and Importance: These results suggest that sepofarsen reinjection intervals may need to be longer than 2 years.

Photoreceptor function and structure in retinal degenerations caused by biallelic BEST1 mutations.

The only approved retinal gene therapy is for biallelic RPE65 mutations which cause a recessive retinopathy with a primary molecular defect located at the retinal pigment epithelium (RPE). For a distinct recessive RPE disease caused by biallelic BEST1 mutations, a pre-clinical proof-of-concept for gene therapy has been demonstrated in canine eyes. The current study was undertaken to consider potential outcome measures for a BEST1 clinical trial in patients demonstrating a classic autosomal recessive bestrophinopathy (ARB) phenotype. Spatial distribution of retinal structure showed a wide expanse of abnormalities including large intraretinal cysts, shallow serous retinal detachments, abnormalities of inner and outer segments, and an unusual prominence of the external limiting membrane. Surrounding the central macula extending from 7 to 30 deg eccentricity, outer nuclear layer was thicker than expected from a cone only retina and implied survival of many rod photoreceptors. Co-localized however, were large losses of rod sensitivity despite preserved cone sensitivities. The dissociation of rod function from rod structure observed, supports a large treatment potential in the paramacular region for biallelic bestrophinopathies.