Three-Year Safety Results of SAR422459 (EIAV-ABCA4) Gene Therapy in Patients With ABCA4-Associated Stargardt Disease: An Open-Label Dose-Escalation Phase I/IIa Clinical Trial, Cohorts 1-5.

PURPOSE: To report on the safety of the first 5 cohorts of a gene therapy trial using recombinant equine infectious anemia virus expressing ABCA4 (EIAV-ABCA4) in adults with Stargardt dystrophy due to mutations in ABCA4. DESIGN: Nonrandomized multicenter phase I/IIa clinical trial. METHODS: Patients received a subretinal injection of EIAVABCA4 in the worse-seeing eye at 3 dose levels and were followed for 3 years after treatment. MAIN OUTCOME MEASURES: The primary end point was ocular and systemic adverse events. The secondary end points were best-corrected visual acuity, static perimetry, kinetic perimetry, total field hill of vision, full field electroretinogram, multifocal ERG, color fundus photography, short-wavelength fundus autofluorescence, and spectral domain optical coherence tomography. RESULTS: The subretinal injections were well tolerated by all 22 patients across 3 dose levels. There was 1 case of a treatment-related ophthalmic serious adverse event in the form of chronic ocular hypertension. The most common adverse events were associated with the surgical procedure. In 1 patient treated with the highest dose, there was a significant decline in the number of macular flecks as compared with the untreated eye. However, in 6 patients, hypoautofluorescent changes were worse in the treated eye than in the untreated eye. Of these, 1 patient had retinal pigment epithelium atrophy that was characteristic of tissue damage likely associated with bleb induction. No patients had any clinically significant changes in best-corrected visual acuity, static perimetry, kinetic perimetry, total field hill of vision, full field electroretinogram, or multifocal ERG attributable to the treatment. CONCLUSIONS: Subretinal treatment with EIAV-ABCA4 was well tolerated with only 1 case of ocular hypertension. No clinically significant changes in visual function tests were found to be attributable to the treatment. However, 27% of treated eyes showed exacerbation of retinal pigment epithelium atrophy on fundus autofluorescence. There was a significant reduction in macular flecks in 1 treated eye from the highest dose cohort. Additional follow-up and continued investigation in more patients will be required to fully characterize the safety and efficacy of EIAV-ABCA4.

Reliability of Spectral-Domain OCT Ellipsoid Zone Area and Shape Measurements in Retinitis Pigmentosa.

PURPOSE: We investigate the ellipsoid zone (EZ) area and EZ boundary shape measurement reliability and the operability characteristics of two methods of EZ boundary delineation in spectral-domain optical coherence tomography (SD-OCT). METHODS: EZ boundaries in SD-OCT scans of 122 eyes from 64 subjects with autosomal dominant retinitis pigmentosa were delineated by three raters using two methods, termed the profile and en face methods. For each method, we determined the measurement reliabilities for boundary area (EZ area) and boundary shape, percentage of eyes with measurable EZ (measurability), time required, and effect of rater experience. RESULTS: With expert raters, inter- and intrarater area intraclass correlation coefficients (ICCs) were 0.986 and 0.980 (profile) and 0.959 and 0.976 (en face), respectively. In comparison, the corresponding shape ICCs were 0.906 and 0.891 (profile) and 0.845 and 0.885 (en face), indicating lower reliability for the raw measurements (P ≤ 0.01). Only profile method interrater reliability depended on experience. Average measurement times per eye were 8.2 (profile) and 4.1 (en face) minutes. Measurability percentages were 99.2% (profile) and 73.0% (en face). CONCLUSIONS: The slower profile method had better measurability, and with expert raters yielded the best area and shape reliabilities. The faster, but less sensitive, en face method still showed excellent reliability, and was less dependent on experience. Shape analysis reveals the boundary measurements underpinning EZ area have lower reliability than suggested by area analysis. TRANSLATIONAL RELEVANCE: This study provides new reliability perspectives and logistical considerations for the manual measurement procedures that generate EZ area outcome measures.

REPEATABILITY AND LONGITUDINAL ASSESSMENT OF FOVEAL CONE STRUCTURE IN CNGB3-ASSOCIATED ACHROMATOPSIA.

PURPOSE: Congenital achromatopsia is an autosomal recessive disease causing substantial reduction or complete absence of cone function. Although believed to be a relatively stationary disorder, questions remain regarding the stability of cone structure over time. In this study, the authors sought to assess the repeatability of and examine longitudinal changes in measurements of central cone structure in patients with achromatopsia. METHODS: Forty-one subjects with CNGB3-associated achromatopsia were imaged over a period of between 6 and 26 months using optical coherence tomography and adaptive optics scanning light ophthalmoscopy. Outer nuclear layer (ONL) thickness, ellipsoid zone (EZ) disruption, and peak foveal cone density were assessed. RESULTS: ONL thickness increased slightly compared with baseline (0.184 µm/month, P = 0.02). The EZ grade remained unchanged for 34/41 subjects. Peak foveal cone density did not significantly change over time (mean change 1% per 6 months, P = 0.126). CONCLUSION: Foveal cone structure showed little or no change in this group of subjects with CNGB3-associated achromatopsia. Over the time scales investigated (6-26 months), achromatopsia seems to be a structurally stable condition, although longer-term follow-up is needed. These data will be useful in assessing foveal cone structure after therapeutic intervention.

Test-Retest Variability of Functional and Structural Parameters in Patients with Stargardt Disease Participating in the SAR422459 Gene Therapy Trial.

PURPOSE: The goal of this analysis was to determine the test-retest variability of functional and structural measures from a cohort of patients with advanced forms of Stargardt Disease (STGD) participating in the SAR422459 (NCT01367444) gene therapy clinical trial. METHODS: Twenty-two participants, aged 24 to 66, diagnosed with advanced forms of STGD, with at least one pathogenic ABCA4 mutation on each chromosome participating in the SAR422459 (NCT01367444) gene therapy clinical trial, were screened over three visits within 3 weeks or less. Functional visual evaluations included: best-corrected visual acuity (BCVA) Early Treatment Diabetic Retinopathy Study (ETDRS) letter score, semiautomated kinetic perimetry (SKP) using isopters I4e, III4e, and V4e, hill of vision (HOV) calculated from static visual fields (SVF) by using a 184n point centrally condensed grid with the stimulus size V test target. Retinal structural changes such as central macular thickness and macular volume were assessed by spectral-domain optical coherence tomography (SD-OCT). Repeatability coefficients (RC) and 95% confidential intervals (CI) were calculated for each parameter using a hierarchical mixed-effects model and bootstrapping. RESULTS: Criteria for statistically significant changes for various parameters were found to be the following: BCVA letter score (8 letters), SKP isopters I4e, III4e, and V4e (3478.85; 2488.02 and 2622.46 deg2, respectively), SVF full volume HOV (VTOT, 14.62 dB-sr), central macular thickness, and macular volume (4.27 µm and 0.15 mm3, respectively). CONCLUSIONS: This analysis provides important information necessary to determine if significant changes are occurring in structural and functional assessments commonly used to measure disease progression in this cohort of patients with STGD. Moreover, this information is useful for future trials assessing safety and efficacy of treatments in STGD. TRANSLATIONAL RELEVANCE: Determination of variability of functional and structural measures in participants with advanced stages of the STGD is necessary to assess efficacy and safety in treatment trials involving STGD patients.

Residual Foveal Cone Structure in CNGB3-Associated Achromatopsia.

PURPOSE: Congenital achromatopsia (ACHM) is an autosomal recessive disorder in which cone function is absent or severely reduced. Gene therapy in animal models of ACHM have shown restoration of cone function, though translation of these results to humans relies, in part, on the presence of viable cone photoreceptors at the time of treatment. Here, we characterized residual cone structure in subjects with CNGB3-associated ACHM. METHODS: High-resolution imaging (optical coherence tomography [OCT] and adaptive optics scanning light ophthalmoscopy [AOSLO]) was performed in 51 subjects with CNGB3-associated ACHM. Peak cone density and inter-cone spacing at the fovea was measured using split-detection AOSLO. Foveal outer nuclear layer thickness was measured in OCT images, and the integrity of the photoreceptor layer was assessed using a previously published OCT grading scheme. RESULTS: Analyzable images of the foveal cones were obtained in 26 of 51 subjects, with nystagmus representing the major obstacle to obtaining high-quality images. Peak foveal cone density ranged from 7,273 to 53,554 cones/mm2, significantly lower than normal (range, 84,733-234,391 cones/mm2), with the remnant cones being either contiguously or sparsely arranged. Peak cone density was correlated with OCT integrity grade; however, there was overlap of the density ranges between OCT grades. CONCLUSIONS: The degree of residual foveal cone structure varies greatly among subjects with CNGB3-associated ACHM. Such measurements may be useful in estimating the therapeutic potential of a given retina, providing affected individuals and physicians with valuable information to more accurately assess the risk-benefit ratio as they consider enrolling in experimental gene therapy trials. (www.clinicaltrials.gov, NCT01846052.).

Results at 2 Years after Gene Therapy for RPE65-Deficient Leber Congenital Amaurosis and Severe Early-Childhood-Onset Retinal Dystrophy.

PURPOSE: To provide an initial assessment of the safety of a recombinant adeno-associated virus vector expressing RPE65 (rAAV2-CB-hRPE65) in adults and children with retinal degeneration caused by RPE65 mutations. DESIGN: Nonrandomized, multicenter clinical trial. PARTICIPANTS: Eight adults and 4 children, 6 to 39 years of age, with Leber congenital amaurosis (LCA) or severe early-childhood-onset retinal degeneration (SECORD). METHODS: Patients received a subretinal injection of rAAV2-CB-hRPE65 in the poorer-seeing eye, at either of 2 dose levels, and were followed up for 2 years after treatment. MAIN OUTCOME MEASURES: The primary safety measures were ocular and nonocular adverse events. Exploratory efficacy measures included changes in best-corrected visual acuity (BCVA), static perimetry central 30° visual field hill of vision (V30) and total visual field hill of vision (VTOT), kinetic perimetry visual field area, and responses to a quality-of-life questionnaire. RESULTS: All patients tolerated subretinal injections and there were no treatment-related serious adverse events. Common adverse events were those associated with the surgical procedure and included subconjunctival hemorrhage in 8 patients and ocular hyperemia in 5 patients. In the treated eye, BCVA increased in 5 patients, V30 increased in 6 patients, VTOT increased in 5 patients, and kinetic visual field area improved in 3 patients. One subject showed a decrease in BCVA and 2 patients showed a decrease in kinetic visual field area. CONCLUSIONS: Treatment with rAAV2-CB-hRPE65 was not associated with serious adverse events, and improvement in 1 or more measures of visual function was observed in 9 of 12 patients. The greatest improvements in visual acuity were observed in younger patients with better baseline visual acuity. Evaluation of more patients and a longer duration of follow-up will be needed to determine the rate of uncommon or rare side effects or safety concerns.

Assessment of Different Sampling Methods for Measuring and Representing Macular Cone Density Using Flood-Illuminated Adaptive Optics.

PURPOSE: To describe a standardized flood-illuminated adaptive optics (AO) imaging protocol suitable for the clinical setting and to assess sampling methods for measuring cone density. METHODS: Cone density was calculated following three measurement protocols: 50 × 50-µm sampling window values every 0.5° along the horizontal and vertical meridians (fixed-interval method), the mean density of expanding 0.5°-wide arcuate areas in the nasal, temporal, superior, and inferior quadrants (arcuate mean method), and the peak cone density of a 50 × 50-µm sampling window within expanding arcuate areas near the meridian (peak density method). Repeated imaging was performed in nine subjects to determine intersession repeatability of cone density. RESULTS: Cone density montages could be created for 67 of the 74 subjects. Image quality was determined to be adequate for automated cone counting for 35 (52%) of the 67 subjects. We found that cone density varied with different sampling methods and regions tested. In the nasal and temporal quadrants, peak density most closely resembled histological data, whereas the arcuate mean and fixed-interval methods tended to underestimate the density compared with histological data. However, in the inferior and superior quadrants, arcuate mean and fixed-interval methods most closely matched histological data, whereas the peak density method overestimated cone density compared with histological data. Intersession repeatability testing showed that repeatability was greatest when sampling by arcuate mean and lowest when sampling by fixed interval. CONCLUSIONS: We show that different methods of sampling can significantly affect cone density measurements. Therefore, care must be taken when interpreting cone density results, even in a normal population.

Long-term characterization of retinal degeneration in rd1 and rd10 mice using spectral domain optical coherence tomography.

PURPOSE: We characterize the in vivo changes over time in the retinal structure of wild-type mice alongside two lines of mice deficient in the ß-subunit of phosphodiesterase (rd1 and rd10 mice) using spectral domain optical coherence tomography (SD-OCT). METHODS: SD-OCT images were obtained using the Bioptigen spectral domain ophthalmic imaging system (SDOIS). Wild-type C57BL/6J, rd1 and rd10 mice ranging in age from P14 to P206 were sedated with 1% isoflurane. Horizontal and vertical linear scans through the optic nerve, and annular scans around the optic nerve were obtained. RESULTS: SD-OCT imaging of wild-type mice demonstrated visibility of the inner segment/outer segment (IS/OS) junction, external limiting membrane (ELM), outer nuclear layer (ONL), and outer plexiform layer (OPL). At P14, most rd10 mice exhibited normal SD-OCT profiles, but some displayed changes in the IS/OS junction. At the same time point, rd1 mice had severe outer retinal degeneration. In rd10 mice, imaging revealed loss of the IS/OS junction by P18, hyperreflective changes in the ONL at P20, hyperreflective vitreous opacities, and shallow separation of the neural retina from the RPE. Retinal separations were not observed in rd1 mice. Segmentation analysis in wild-type mice demonstrated relatively little variability between animals, while in rd10 and rd1 mice there was a steady decline in outer retinal thickness. Histologic studies demonstrated correlation of retinal features with those seen on SD-OCT scans. Segmentation analysis provides a quantitative and reproducible method for measuring in vivo retinal changes in mice. CONCLUSIONS: SD-OCT provides a non-invasive method of following long-term retinal changes in mice in vivo. Although rd10 and rd1 mice have mutations in the same gene, they demonstrate significantly different features on SD-OCT.