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The purpose of this study was to evaluate rod and cone function and outer retinal structure within macular lesions, and surrounding extralesional areas of patients with autosomal dominant Best vitelliform macular dystrophy caused by BEST1 mutations.Seventeen patients from seven families were examined with dark- and light-adapted chromatic perimetry and optical coherence tomography. Subsets of patients had long-term follow-up (14-22 years, n = 6) and dark-adaptation kinetics measured (n = 5).Within central lesions with large serous retinal detachments, rod sensitivity was severely reduced but visual acuity and cone sensitivity were relatively retained. In surrounding extralesional areas, there was a mild but detectable widening of the subretinal space in some patients and some retinal areas. Available evidence was consistent with subretinal widening causing slower dark-adaptation kinetics. Over long-term follow-up, some eyes showed formation of de novo satellite lesions at retinal locations that years previously demonstrated subretinal widening. A subclinical abnormality consisting of a retina-wide mild thickening of the outer nuclear layer was evident in many patients and thickening increased in the subset of patients with long-term follow-up.Outcome measures for future clinical trials should include evaluations of rod sensitivity within central lesions and quantitative measures of outer retinal structure in normal-appearing regions surrounding the lesions.

Leber congenital amaurosis due to CEP290 ciliopathy is being explored by treatment with an antisense oligonucleotide (AON) sepofarsen. One individual who was part of a larger cohort (ClinicalTrials.gov no. NCT03140969) was studied for 15 months after a single intravitreal sepofarsen injection. Concordant measures of visual function and retinal structure reached a substantial efficacy peak near 3 months post-injection. At 15 months, there was sustained efficacy even though there was evidence of reduction from peak response. Efficacy kinetics can be explained by the balance of AON-driven new CEP290 protein synthesis and a slow natural rate of CEP290 protein degradation in human foveal cone photoreceptors.

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.

CEP290-associated Leber congenital amaurosis type 10 (LCA10) is a retinal disease resulting in childhood blindness. Sepofarsen is an RNA antisense oligonucleotide targeting the c.2991+1655A>G variant in the CEP290 gene to treat LCA10. In this open-label, phase 1b/2 (NCT03140969), 12-month, multicenter, multiple-dose, dose-escalation trial, six adult patients and five pediatric patients received ≤4 doses of intravitreal sepofarsen into the worse-seeing eye. The primary objective was to evaluate sepofarsen safety and tolerability via the frequency and severity of ocular adverse events (AEs); secondary objectives were to evaluate pharmacokinetics and efficacy via changes in functional outcomes. Six patients received sepofarsen 160 µg/80 µg, and five patients received sepofarsen 320 µg/160 µg. Ten of 11 (90.9%) patients developed ocular AEs in the treated eye (5/6 with 160 µg/80 µg; 5/5 with 320 µg/160 µg) versus one of 11 (9.1%) in the untreated eye; most were mild in severity and dose dependent. Eight patients developed cataracts, of which six (75.0%) were categorized as serious (2/3 with 160 µg/80 µg; 4/5 with 320 µg/160 µg), as lens replacement was required. As the 160-µg/80-µg group showed a better benefit–risk profile, higher doses were discontinued or not initiated. Statistically significant improvements in visual acuity and retinal sensitivity were reported (post hoc analysis). The manageable safety profile and improvements reported in this trial support the continuation of sepofarsen development.

Summary A first-in-human clinical trial of gene therapy in Leber congenital amaurosis due to mutations in the GUCY2D gene is underway, and early results are summarized. A recombinant adeno-associated virus serotype 5 (rAAV5) vector carrying the human GUCY2D gene was delivered by subretinal injection to one eye in three adult patients with severe visual loss, nystagmus, but preserved retinal structure. Safety and efficacy parameters were monitored for 9 months post-operatively. No systemic toxicity was detected; there were no serious adverse events, and ocular adverse events resolved. P1 and P2 showed statistically significant rod photoreceptor vision improvement by full-field stimulus testing in the treated eye. P1 also showed improvement in pupillary responses. Visual acuity remained stable from baseline in P1 and P2. P3, however, showed a gain of 0.3 logMAR in the treated eye, indicating greater cone-photoreceptor function. The results show safety and both rod- and cone-mediated efficacy of this therapy., Graphical abstract, Highlights • Blindness from genetic disorders of the retina has been incurable for centuries • The first photoreceptor-based childhood blindness (GUCY2D-LCA) has now been treated • Proof of safety and efficacy of subretinal gene therapy in GUCY2D-LCA is reported, Clinical Genetics ; Clinical Finding ; Techniques in Genetics

Photoreceptor ciliopathies constitute the most common molecular mechanism of the childhood blindness Leber congenital amaurosis. Ten patients with Leber congenital amaurosis carrying the c.2991+1655A>G allele in the ciliopathy gene centrosomal protein 290 (CEP290) were treated (ClinicalTrials.gov no. NCT03140969 ) with intravitreal injections of an antisense oligonucleotide to restore correct splicing. There were no serious adverse events, and vision improved at 3 months. The visual acuity of one exceptional responder improved from light perception to 20/400. RNA antisense oligonucleotide therapy to restore normal splicing of a ciliopathy gene shows promising safety and efficacy results in a clinical trial to treat a form of childhood blindness.

Inherited retinal diseases (IRDs) were first classified clinically by history, ophthalmoscopic appearance, type of visual field defects, and electroretinography (ERG). ERGs isolating the two major photoreceptor types (rods and cones) showed some IRDs with greater cone than rod retinal dysfunction; others were the opposite. Within the cone-rod diseases, there can be phenotypic variability, which can be attributed to genetic heterogeneity and the variety of visual function mechanisms that are disrupted. Most cause symptoms from childhood or adolescence, although others can manifest later in life. Among the causative genes for cone-rod dystrophy (CORD) are those encoding molecules in phototransduction cascade activation and recovery processes, photoreceptor outer segment structure, the visual cycle and photoreceptor development. We review 11 genes known to cause cone-rod disease in the context of their roles in normal visual function and retinal structure. Knowledge of the pathobiology of these genetic diseases is beginning to pave paths to therapy.

Disease mechanisms have become better understood in previously incurable forms of early-onset severe retinal dystrophy, such as Leber congenital amaurosis (LCA). This has led to novel treatments and clinical trials that have shown some success. Standard methods to measure vision were difficult if not impossible to perform in severely affected patients with low vision and nystagmus. To meet the need for visual assays, we devised a psychophysical method, which we named full-field stimulus testing (FST). From early versions based on an automated perimeter, we advanced FST to a more available light-emitting diode platform. The journey from invention to use of such a technique in our inherited retinal degeneration clinic is reviewed and many of the lessons learned over the 15 years of application of FST are explained. Although the original purpose and application of FST was to quantify visual thresholds in LCA, there are rare opportunities for FST also to be used beyond LCA to measure aspects of vision in other inherited retinal degenerations; examples are given. The main goal of the current review, however, remains to enable investigators studying and treating LCA to understand how to best use FST and how to reduce artefact and confounding complexities so the test results become more valuable to the understanding of LCA diseases and results of novel interventions.

Background: Sepofarsen is an RNA antisense oligonucleotide (AON) targeting the common c.2991+1655A>G mutation in the CEP290 gene to treat Leber congenital amaurosis type 10 (LCA10), a condition with severe childhood-onset vision loss or blindness. Methods: In this 12-month, multicenter, open-label, multiple-dose, dose-escalation, phase 1b/2 trial, patients with LCA10 received up to four doses of intravitreal sepofarsen in the worse-seeing eye. Primary end point was safety; secondary end points included vision outcome measures. Findings: Of 11 patients enrolled (8–44 years of age), six received loading/maintenance doses of sepofarsen of 160 µg/80 µg and five received 320 µg/160 µg. Reported cases of cataracts, mild cystoid macular edema, and retinal thinning were 3, 0, and 0, respectively, in the 160-µg/80-µg group and 5, 2, and 2 in the 320-µg/160-µg group. Pooled data (n=11) showed improvements from baseline to Month 12 in treated eyes vs untreated eyes in mean ± standard error of the mean best-corrected visual acuity (BCVA: −0·55 ± 0·26 vs −0·12 ± 0·07 logarithm of the minimum angle of resolution [logMAR], p

Purpose To use supervised machine learning to predict visual function from retinal structure in retinitis pigmentosa (RP) and apply these estimates to CEP290- and NPHP5-associated Leber congenital amaurosis (LCA) to determine the potential for functional improvement. Methods Patients with RP (n = 20) and LCA due to CEP290 (n = 12) or NPHP5 (n = 6) mutations were studied. A patient with CEP290 mutations but mild retinal degeneration was included. RP patients had cone-mediated macular function. A machine learning technique was used to associate perimetric sensitivities to local structure in RP patients. Models trained on RP data were applied to predict visual function in LCA. Results The RP and LCA patients had comparable retinal structure. RP patients had peak sensitivity at the fovea surrounded by decreasing sensitivity. Machine learning could successfully predict perimetry results from segmented or unsegmented optical coherence tomography (OCT) input. Application of machine learning predictions to LCA within the residual macular island of photoreceptor structure showed differences between predicted and measured sensitivities defining treatment potential. In patients with retained vision, the treatment potential was 4.6 ± 2.9 dB at the fovea but 16.4 ± 4.4 dB at the parafovea. In patients with limited or no vision, the treatment potential was 17.6 ± 9.4 dB. Conclusions Cone vision improvement potential in LCA due to CEP290 or NPHP5 mutations is predictable from retinal structure using a machine learning approach. This should allow individual prediction of the maximal efficacy in clinical trials and guide decisions about dosing. Similar strategies can be used in other retinal degenerations to estimate the extent and location of treatment potential.

Due to improved phenotyping and genetic characterization, the field of ‘incurable’ and ‘blinding’ inherited retinal diseases (IRDs) has moved substantially forward. Decades of ascertainment of IRD patient data from Philadelphia and Toronto centers illustrate the progress from Mendelian genetic types to molecular diagnoses. Molecular genetics have been used not only to clarify diagnoses and to direct counseling but also to enable the first clinical trials of gene-based treatment in these diseases. An overview of the recent reports of gene augmentation clinical trials by subretinal injections is used to reflect on the reasons why there has been limited success in this early venture into therapy. These first-in human experiences have taught that there is a need for advancing the techniques of delivery of the gene products - not only for refining further subretinal trials, but also for evaluating intravitreal delivery. Candidate IRDs for intravitreal gene delivery are then suggested to illustrate some of the disorders that may be amenable to improvement of remaining central vision with the least photoreceptor trauma. A more detailed understanding of the human IRDs to be considered for therapy and the calculated potential for efficacy should be among the routine prerequisites for initiating a clinical trial.

Purpose To determine the change in vision and retinal structure in patients with the common c.2299delG mutation in the USH2A gene in anticipation of clinical trials of therapy. Design Retrospective observational case series. Methods Eighteen patients, homozygotes or compound heterozygotes with the c.2299delG mutation in USH2A, were studied with regard to visual acuity, kinetic perimetry, dark- and light-adapted static perimetry, optical coherence tomography (OCT), and autofluorescence (AF) imaging. Serial data were available for at least half of the patients, depending on the parameter analyzed. Results The kinetics of disease progression in this specific molecular form of USH2A differed between the measured parameters. Visual acuity could remain normal for decades. Kinetic and light-adapted static perimetry across the entire visual field had similar rates of decline that were slower than those of rod-based perimetry. Horizontal OCT scans through the macula showed that inner segment/outer segment line width had a similar rate of constriction as co-localized AF imaging and cone-based light-adapted sensitivity extent. The rate of constriction of rod-based sensitivity extent across this same region was twice as rapid as that of cones. Conclusions In patients with the c.2299delG mutation in USH2A, rod photoreceptors are the cells that express disease early and more aggressively than cones. Rod-based vision measurements in central or extracentral-peripheral retinal regions warrant monitoring in order to complete a clinical trial in a timely manner.

Mutations in the EYS (eyes shut homolog) gene are a common cause of autosomal recessive (ar) retinitis pigmentosa (RP). Without a mammalian model of human EYS disease, there is limited understanding of details of disease expression and rates of progression of the retinal degeneration. We studied clinically and with chromatic static perimetry, spectral-domain optical coherence tomography (OCT), and en face autofluoresence imaging, a cohort of 15 patients (ages 12–51 at first visit), some of whom had longitudinal data of function and structure. Rod sensitivity was able to be measured by chromatic perimetry in most patients at their earliest visits and some patients retained patchy rod function into the fifth decade of life. As expected from RP, cone sensitivity persisted after rod function was no longer measurable. The photoreceptor nuclear layer of the central retina was abnormal except at the fovea in most patients at first visit. Perifoveal disease measured over a period of years indicated that photoreceptor structural loss was followed by dysmorphology of the inner retina and loss of retinal pigment epithelial integrity. Although there could be variability in severity, preliminary analyses of the rates of vision loss suggested that EYS is a more rapidly progressive disease than other ciliopathies causing arRP, such as USH2A and MAK.

Recessively-inherited NR2E3 gene mutations cause an unusual retinopathy with abnormally-increased short-wavelength sensitive cone (S-cone) function, in addition to reduced rod and long/middle-wavelength sensitive cone (L/M-cone) function. Progress toward clinical trials to treat patients with this otherwise incurable retinal degeneration prompted the need to determine efficacy outcome measures. Comparisons were made between three computerized perimeters available in the clinic. These perimeters could deliver short-wavelength stimuli on longer-wavelength adapting backgrounds to measure whether S-cone vision can be quantified. Results from a cohort of normal subjects were compared across the three perimeters to determine S-cone isolation and test-retest variability. S-cone perimetry data from NR2E3-ESCS (enhanced S-cone syndrome) patients were examined and determined to have five stages of disease severity. Using these stages, strategies were proposed for monitoring efficacy of either a focal or retina-wide intervention. This work sets the stage for clinical trials.

Purpose To determine the progression rate and the variability of rod and cone sensitivities in patients with X-linked retinitis pigmentosa (XLRP) caused by mutations in ORF15-RPGR. Methods ORF15-RPGR-XLRP patients (n = 15) were studied prospectively over 2 years with static perimetry sampling the visual field under dark-adapted and light-adapted conditions on a 12° square grid covering 168° width and 84° height. Natural history of rod and cone sensitivity loss and test-retest variability were estimated. Data were analyzed pointwise as well as averaged across small regions of neighboring loci of approximately 80 mm2 (900 deg2) in size representing the likely extent of localized gene therapy injections. Results Retinal loci with mild to moderate loss of sensitivity tended to be in the mid- to far-peripheral retina in most patients. When averaged across small regions, dark-adapted rod vision progressed at an average of 2 dB per year with a coefficient of repeatability (CR) of 6.3 dB, and light-adapted cone vision with white stimulus progressed at an average of 0.9 dB per year with a CR of 3.8 dB. For an average patient enrolled in an early-phase clinical trial, significant (α = 0.05) progression would be predicted to occur with 80% power in 4.5 years for rod vision and 6.1 years for cone vision. Localization of regions in the temporal hemifield and grouping of results from multiple patients would permit trial designs of shorter duration. Conclusions Measurement of rod sensitivity under dark-adapted conditions averaged across a small region showed the greatest potential for detectability of progression in the shortest period.

Purpose To determine the progression of cone vision loss in patients with recessive disease from NR2E3 gene mutations. Methods Patients with NR2E3 mutations (n = 37) were studied as a retrospective observational case series clinically and with chromatic static perimetry. Patients were investigated cross-sectionally, and a subset was followed longitudinally. Results Patients showed a range of visual acuities; there was no clear relationship to age. With kinetic perimetry (V4e target), a full field could be retained over many years. Other patients showed progression from a full field, with or without pericentral scotomas, to a small central island. Three patterns of S-cone function were defined, based on percentage of hypersensitive S-cone loci in the field. From occupying most of the visual field, hyperfunctioning S-cone loci could diminish in percent, remaining largely in the periphery. Normal S-cone functioning then dominates, followed by the appearance of an annular region of abnormal S-cone loci approximately 10° to 40° from the fovea. Overall, S-cone sensitivity declined 2.6 times faster than L/M-cone sensitivity. Conclusions Murine proof-of-concept studies suggest that clinical trials of patients with NR2E3 mutations may be forthcoming. Patterns of S-cone hyperfunction across the field would serve as a means to categorize patients as entry criteria or cohort selection in clinical trials. S-cone perimetry can be measured in the clinic and would be the logical efficacy monitor for therapeutic strategies. Given further understanding of the natural history of the disease, targeting the annular region of S-cone dysfunction for a focal therapy or for monitoring in a retina-wide intervention warrants consideration.