Introduction: Inherited retinal degenerations (IRDs) have long been considered untreatable and incurable. Recently, one form of early-onset autosomal recessive IRD, Leber congenital amaurosis (LCA) caused by mutations in RPE65 (retinal pigment epithelium-specific protein 65 kDa) gene, has responded with some improvement of vision to gene augmentation therapy and oral retinoid administration. This early success now requires refinement of such therapeutics to fully realize the impact of these major scientific and clinical advances. Areas covered: Progress toward human therapy for RPE65-LCA is detailed from the understanding of molecular mechanisms to preclinical proof-of-concept research to clinical trials. Unexpected positive and complicating results in the patients receiving treatment are explained. Logical next steps to advance the clinical value of the therapeutics are suggested. Expert opinion: The first molecularly based early-phase therapies for an IRD are remarkably successful in that vision has improved and adverse events are mainly associated with surgical delivery to the subretinal space. Yet, there are features of the gene augmentation therapeutic response, such as slowed kinetics of night vision, lack of foveal cone function improvement and relentlessly progressive retinal degeneration despite therapy, that still require research attention.

More than 100 different mutations in the RPE65 gene are associated with inherited retinal degeneration. Although some missense mutations have been shown to abolish isomerase activity of RPE65, the molecular bases leading to loss of function and retinal degeneration remain incompletely understood. Here we show that several missense mutations resulted in significant decrease in expression level of RPE65 in the human retinal pigment epithelium cells. The 26S proteasome non-ATPase regulatory subunit 13, a newly identified negative regulator of RPE65, mediated degradation of mutant RPE65s, which were misfolded and formed aggregates in the cells. Many mutations, including L22P, T101I, and L408P, were mapped on nonactive sites of RPE65. Enzyme activities of these mutant RPE65s were significantly rescued at low temperature, whereas mutant RPE65s with a distinct active site mutation could not be rescued under the same conditions. 4-phenylbutyrate (PBA) displayed a significant synergistic effect on the low temperature-mediated rescue of the mutant RPE65s. Our results suggest that a low temperature eye mask and PBA, a FDA-approved oral medicine, may provide a promising "protein repair therapy" that can enhance the efficacy of gene therapy for delaying retinal degeneration caused by RPE65 mutations.

Purpose

SP4 is a transcription factor abundantly expressed in retina that binds to the GC promoter region of photoreceptor signal transduction genes. We have previously shown that SP4 may be involved in the transcriptional activation of these genes alone or together with other transcription factors such as SP1, neural retina leucine zipper protein (NRL), and cone-rod homeobox gene (CRX). Since mutations in NRL and CRX are involved in inherited retinal degenerations, SP4 was considered a good candidate for mutation screening in patients with this type of diseases. The purpose of this work, therefore, was to investigate possible mutations in SP4 in a cohort of patients affected with different forms of retinal degenerations.

Methods

270 unrelated probands with various forms of retinal degeneration including autosomal dominant and autosomal recessive retinitis pigmentosa (RP), autosomal dominant and autosomal recessive cone-rod dystrophy (CRD), and Leber's congenital amaurosis (LCA), were screened for mutations in the SP4 gene. Single strand conformation polymorphism (SSCP) analysis was performed on the six SP4 gene exons including flanking regions followed by direct sequencing of SSCP variants.

Results

Nine different sequence variants were found in 29 patients, four in introns and five in exons. Many of the probands were previously screened for mutations in the genes encoding the alpha-, beta- and gamma-subunits of rod-specific cGMP phosphodiesterase (PDE6A, PDE6B, PDE6G), the beta-subunit of rod-specific transducin (GNB1), and peripherin/rds (RDS). One group of seven probands of Hispanic background that included five with arRP, one with RP of unknown inheritance (isolate) and 1 with arCRD carried an Asn306Ser mutation in SP4. Of the seven, the isolate case was homozygous and the other 6 heterozygous for the variant. Two arRP and the arCRD probands carried an additional intronic GNB1 variant. DNA from the family members of the arCRD proband could not be obtained, but for the other two families, all affected members and none of the unaffected carried both the SP4 Asn306Ser allele and the GNB1 intronic variant.

Conclusions

If mutations in SP4 do cause retinal degenerative disease, their frequency would be low. While digenic disease with the SP4 Asn306Ser and the GNB1 intronic variant alleles has not been established, neither has it been ruled out. This leaves open the possibility of a cooperative involvement of SP4 and GNB1 in the normal function of the retina.

The following review summarizes the state of the art in representative aspects of gene therapy/translational medicine and evolves from a symposium held at the School of Veterinary Medicine, University of Pennsylvania on November 16, 2017 honoring Dr. Gustavo Aguirre, recipient of ARVO's 2017 Proctor Medal. Focusing on the retina, speakers highlighted current work on moving therapies for inherited retinal degenerative diseases from the laboratory bench to the clinic.

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.

Background

Mutations in the peripherin/retinal degeneration slow (RDS) gene have been identified in patients with retinitis pigmentosa and pattern macular dystrophy. The authors initially examined a large family affected with both peripheral and macular degeneration, inherited as an autosomal dominant trait. Screening for peripherin/RDS mutations identified a previously unreported nucleotide alteration in all of the affected individuals. Two additional families later were found to have this same mutation.

Methods

DNA samples from the members of three unrelated families were screened for peripherin/RDS mutations by denaturing gradient gel electrophoresis of the polymerase chain reaction-amplified peripherin/RDS coding sequences. The sequence change that was detected was further characterized by DNA sequencing. Family members were examined and evaluated with psychophysical and electrophysiologic methods.

Results

A proline to arginine mutation in codon 210 of peripherin/RDS was found in all clinically affected individuals. Macular changes included extensive geographic atrophy, pigment epithelial changes, and/or drusen. The proline to arginine mutation was not found among 100 healthy individuals, making it unlikely to be a nondisease-causing polymorphism.

Conclusions

The authors identified a novel peripherin/RDS gene mutation associated with autosomal dominant retinal degeneration in patients from three different families. The largest family showed a broad variability in the expressivity of the mutation. The overlap of clinical features with those of age-related maculopathy highlights the need to consider photoreceptor-specific genes as potential factors in the etiology of the latter condition.

Mutations in the MYO7A gene cause a deaf-blindness disorder, known as Usher syndrome 1B.  In the retina, the majority of MYO7A is in the retinal pigmented epithelium (RPE), where many of the reactions of the visual retinoid cycle take place.  We have observed that the retinas of Myo7a-mutant mice are resistant to acute light damage. In exploring the basis of this resistance, we found that Myo7a-mutant mice have lower levels of RPE65, the RPE isomerase that has a key role in the retinoid cycle.  We show for the first time that RPE65 normally undergoes a light-dependent translocation to become more concentrated in the central region of the RPE cells.  This translocation requires MYO7A, so that, in Myo7a-mutant mice, RPE65 is partly mislocalized in the light.  RPE65 is degraded more quickly in Myo7a-mutant mice, perhaps due to its mislocalization, providing a plausible explanation for its lower levels.  Following a 50-60% photobleach, Myo7a-mutant retinas exhibited increased all-trans-retinyl ester levels during the initial stages of dark recovery, consistent with a deficiency in RPE65 activity.  Lastly, MYO7A and RPE65 were co-immunoprecipitated from RPE cell lysate by antibodies against either of the proteins, and the two proteins were partly colocalized, suggesting a direct or indirect interaction.  Together, the results support a role for MYO7A in the translocation of RPE65, illustrating the involvement of a molecular motor in the spatiotemporal organization of the retinoid cycle in vision.

Human X-linked blue-cone monochromacy (BCM), a disabling congenital visual disorder of cone photoreceptors, is a candidate disease for gene augmentation therapy. BCM is caused by either mutations in the red (OPN1LW) and green (OPN1MW) cone photoreceptor opsin gene array or large deletions encompassing portions of the gene array and upstream regulatory sequences that would predict a lack of red or green opsin expression. The fate of opsin-deficient cone cells is unknown. We know that rod opsin null mutant mice show rapid postnatal death of rod photoreceptors. Using in vivo histology with high-resolution retinal imaging, we studied a cohort of 20 BCM patients (age range 5-58) with large deletions in the red/green opsin gene array. Already in the first years of life, retinal structure was not normal: there was partial loss of photoreceptors across the central retina. Remaining cone cells had detectable outer segments that were abnormally shortened. Adaptive optics imaging confirmed the existence of inner segments at a spatial density greater than that expected for the residual blue cones. The evidence indicates that human cones in patients with deletions in the red/green opsin gene array can survive in reduced numbers with limited outer segment material, suggesting potential value of gene therapy for BCM.

Purpose

The purpose of this study was to compare the natural history of visual function change in cohorts of patients affected with retinal degeneration due to biallelic variants in Bardet-Biedl syndrome genes: BBS1 and BBS10.

Methods

Patients were recruited from nine academic centers from six countries (Belgium, Canada, France, New Zealand, Switzerland, and the United States). Inclusion criteria were: (1) female or male patients with a clinical diagnosis of retinal dystrophy, (2) biallelic disease-causing variants in BBS1 or BBS10, and (3) measures of visual function for at least one visit. Retrospective data collected included genotypes, age, onset of symptoms, and best corrected visual acuity (VA). When possible, data on refractive error, fundus images and autofluorescence (FAF), optical coherence tomography (OCT), Goldmann kinetic perimetry (VF), electroretinography (ERG), and the systemic phenotype were collected.

Results

Sixty-seven individuals had variants in BBS1 (n = 38; 20 female patients and 18 male patients); or BBS10 (n = 29; 14 female patients and 15 male patients). Missense variants were the most common type of variants for patients with BBS1, whereas frameshift variants were most common for BBS10. When ERGs were recordable, rod-cone dystrophy (RCD) was observed in 82% (23/28) of patients with BBS1 and 73% (8/11) of patients with BBS10; cone-rod dystrophy (CORD) was seen in 18% of patients with BBS1 only, and cone dystrophy (COD) was only seen in 3 patients with BBS10 (27%). ERGs were nondetectable earlier in patients with BBS10 than in patients with BBS1. Similarly, VA and VF declined more rapidly in patients with BBS10 compared to patients with BBS1.

Conclusions

Retinal degeneration appears earlier and is more severe in BBS10 cases as compared to those with BBS1 variants. The course of change of visual function appears to relate to genetic subtypes of BBS.

Purpose

To determine the proportion of male patients presenting simplex retinal degenerative disease (RD: retinitis pigmentosa [RP] or cone/cone-rod dystrophy [COD/CORD]) with mutations in the X-linked retinal degeneration genes RPGR and RP2.

Methods

Simplex males were defined as patients with no known affected family members. Patients were excluded if they had a family history of parental consanguinity. Blood samples from a total of 214 simplex males with a diagnosis of retinal degeneration were collected for genetic analysis. The patients were screened for mutations in RPGR and RP2 by direct sequencing of PCR-amplified genomic DNA.

Results

We identified pathogenic mutations in 32 of the 214 patients screened (15%). Of the 29 patients with a diagnosis of COD/CORD, four mutations were identified in the ORF15 mutational hotspot of the RPGR gene. Of the 185 RP patients, three patients had mutations in RP2 and 25 had RPGR mutations (including 12 in the ORF15 region).

Conclusions

This study represents mutation screening of RPGR and RP2 in the largest cohort, to date, of simplex males affected with RP or COD/CORD. Our results demonstrate a substantial contribution of RPGR mutations to retinal degenerations, and in particular, to simplex RP. Based on our findings, we suggest that RPGR should be considered as a first tier gene for screening isolated males with retinal degeneration.