Adeno-Associated Virus Serotype 2-hCHM Subretinal Delivery to the Macula in Choroideremia: Two-Year Interim Results of an Ongoing Phase I/II Gene Therapy Trial.

PURPOSE: To assess the safety of the subretinal delivery of a recombinant adeno-associated virus serotype 2 (AAV2) vector carrying a human choroideremia (CHM)-encoding cDNA in CHM. DESIGN: Prospective, open-label, nonrandomized, dose-escalation, phase I/II clinical trial. PARTICIPANTS: Fifteen CHM patients (ages 20-57 years at dosing). METHODS: Patients received uniocular subfoveal injections of low-dose (up to 5 × 1010 vector genome [vg] per eye, n = 5) or high-dose (up to 1 × 1011 vg per eye, n = 10) of a recombinant adeno-associated virus serotype 2 (AAV2) vector carrying a human CHM-encoding cDNA (AAV2-hCHM). Patients were evaluated preoperatively and postoperatively for 2 years with ophthalmic examinations, multimodal retinal imaging, and psychophysical testing. MAIN OUTCOME MEASURES: Visual acuity, perimetry (10-2 protocol), spectral-domain OCT (SD-OCT), and short-wavelength fundus autofluorescence (SW-FAF). RESULTS: We detected no vector-related or systemic toxicities. Visual acuity returned to within 15 letters of baseline in all but 2 patients (1 developed acute foveal thinning, and 1 developed a macular hole); the rest showed no gross changes in foveal structure at 2 years. There were no significant differences between intervention and control eyes in mean light-adapted sensitivity by perimetry or in the lateral extent of retinal pigment epithelium relative preservation by SD-OCT and SW-FAF. Microperimetry showed nonsignificant (< 3 standard deviations of the intervisit variability) gains in sensitivity in some locations and participants in the intervention eye. There were no obvious dose-dependent relationships. CONCLUSIONS: Visual acuity was within 15 letters of baseline after the subfoveal AAV2-hCHM injections in 13 of 15 patients. Acute foveal thinning with unchanged perifoveal function in 1 patient and macular hole in 1 patient suggest foveal vulnerability to the subretinal injections. Longer observation intervals will help establish the significance of the minor differences in sensitivities and rate of disease progression observed between intervention and control eyes.

Natural History of the Central Structural Abnormalities in Choroideremia: A Prospective Cross-Sectional Study.

PURPOSE: To describe in detail the central retinal structure of a large group of patients with choroideremia (CHM). DESIGN: A prospective, cross-sectional, descriptive study. PARTICIPANTS: Patients (n = 97, age 6-71 years) with CHM and subjects with normal vision (n = 44; ages 10-50 years) were included. METHODS: Subjects were examined with spectral-domain optical coherence tomography (SD OCT) and near-infrared reflectance imaging. Visual acuity (VA) was measured during their encounter or obtained from recent ophthalmic examinations. Visual thresholds were measured in a subset of patients (n = 24) with automated static perimetry within the central regions (±15°) examined with SD OCT. MAIN OUTCOME MEASURES: Visual acuity and visual thresholds; total nuclear layer, inner nuclear layer (INL), and outer nuclear layer (ONL) thicknesses; and horizontal extent of the ONL and the photoreceptor outer segment (POS) interdigitation zone (IZ). RESULTS: Earliest abnormalities in regions with normally appearing retinal pigment epithelium (RPE) were the loss of the POS and ellipsoid zone associated with rod dysfunction. Transition zones (TZs) from relatively preserved retina to severe ONL thinning and inner retinal thickening moved centripetally with age. Most patients (88%) retained VAs better than 20/40 until their fifth decade of life. The VA decline coincided with migration of the TZ near the foveal center. There were outer retinal tubulations in degenerated, nonatrophic retina in the majority (69%) of patients. In general, RPE abnormalities paralleled photoreceptor degeneration, although there were regions with detectable but abnormally thin ONL co-localizing with severe RPE depigmentation and choroidal thinning. CONCLUSIONS: Abnormalities of the POS and rod dysfunction are the earliest central abnormalities observed in CHM. Foveal function is relatively preserved until the fifth decade of life. Migration of the TZs to the foveal center with foveal thinning and structural disorganization heralded central VA loss. The relationships established may help outline the eligibility criteria and outcome measures for clinical trials for CHM.

Short-term Assessment of Subfoveal Injection of Adeno-Associated Virus-Mediated hCHM Gene Augmentation in Choroideremia Using Adaptive Optics Ophthalmoscopy.

Importance: Subretinal injection for gene augmentation in retinal degenerations forcefully detaches the neural retina from the retinal pigment epithelium, potentially damaging photoreceptors and/or retinal pigment epithelium cells. Objective: To use adaptive optics scanning light ophthalmoscopy (AOSLO) to assess the short-term integrity of the cone mosaic following subretinal injections of adeno-associated virus vector designed to deliver a functional version of the CHM gene (AAV2-hCHM) in patients with choroideremia. Design, Setting, and Participants: This longitudinal case series study enrolled adult patients with choroideremia from February 2015 to January 2016 in the US. To be included in the study, study participants must have received uniocular subfoveal injections of low-dose (5 × 1010 vector genome per eye) or high-dose (1 × 1011 vector genome per eye) AAV2-hCHM. Analysis began February 2015. Main Outcomes and Measures: The macular regions of both eyes were imaged before and 1 month after injection using a custom-built multimodal AOSLO. Postinjection cone inner segment mosaics were compared with preinjection mosaics at multiple regions of interest. Colocalized spectral-domain optical coherence tomography and dark-adapted cone sensitivity was also acquired at each time point. Results: Nine study participants ranged in age from 26 to 50 years at the time of enrollment, and all were White men. Postinjection AOSLO images showed preservation of the cone mosaic in all 9 AAV2-hCHM-injected eyes. Mosaics appeared intact and contiguous 1 month postinjection, with the exception of foveal disruption in 1 patient. Optical coherence tomography showed foveal cone outer segment shortening postinjection. Cone-mediated sensitivities were unchanged in 8 of 9 injected and 9 of 9 uninjected eyes. One participant showed acute loss of foveal optical coherence tomography cone outer segment-related signals along with cone sensitivity loss that colocalized with disruption of the mosaic on AOSLO. Conclusions and Relevance: Integrity of the cone mosaic is maintained following subretinal delivery of AAV2-hCHM, providing strong evidence in support of the safety of the injections. Minor foveal thinning observed following surgery corresponds with short-term cone outer segment shortening rather than cone cell loss. Foveal cone loss in 1 participant raises the possibility of individual vulnerability to the subretinal injection.

Treatment Potential for LCA5-Associated Leber Congenital Amaurosis.

Purpose: To determine the therapeutic window for gene augmentation for Leber congenital amaurosis (LCA) associated with mutations in LCA5. Methods: Five patients (ages 6-31) with LCA and biallelic LCA5 mutations underwent an ophthalmic examination including optical coherence tomography (SD-OCT), full-field stimulus testing (FST), and pupillometry. The time course of photoreceptor degeneration in the Lca5gt/gt mouse model and the efficacy of subretinal gene augmentation therapy with AAV8-hLCA5 delivered at postnatal day 5 (P5) (early, n = 11 eyes), P15 (mid, n = 14), and P30 (late, n = 13) were assessed using SD-OCT, histologic study, electroretinography (ERG), and pupillometry. Comparisons were made with the human disease. Results: Patients with LCA5-LCA showed a maculopathy with detectable outer nuclear layer (ONL) in the pericentral retina and at least 4 log units of dark-adapted sensitivity loss. The Lca5gt/gt mouse has a similarly severe and rapid photoreceptor degeneration. The ONL became progressively thinner and was undetectable by P60. Rod- and cone-mediated ERGs were severely reduced in amplitudes at P30 and became nondetectable by P60. Subretinal AAV8-hLCA5 administered to Lca5gt/gt mice at P5 and P15, but not at P30, resulted in structural and functional rescue. Conclusions: LCA5-LCA is a particularly severe form of LCA that was recapitulated in the Lca5gt/gt mouse. Gene augmentation resulted in structural and functional rescue in the Lca5gt/gt mouse if delivered before P30. Retained photoreceptors were visible within the central retina in all patients with LCA5-LCA, at a level equivalent to that observed in rescued Lca5gt/gt mice, suggesting a window of opportunity for the treatment of patients with LCA5-LCA.

Comparing Clinical Perimetry and Population Receptive Field Measures in Patients with Choroideremia.

Purpose: Choroideremia (CHM) is an X-linked recessive form of hereditary retinal degeneration, which, at advanced stages, leaves only small central islands of preserved retinal tissue. Unlike many other retinal diseases, the spared tissue in CHM supports excellent central vision and stable fixation. Such spared topography in CHM presents an ideal platform to explore the relationship between preserved central retinal structure and the retinotopic organization of visual cortex by using functional magnetic resonance imaging (fMRI). Methods: fMRI was conducted in four participants with CHM and four healthy control participants while they viewed drifting contrast pattern stimuli monocularly. A single ∼3-minute fMRI run was collected for each eye separately. fMRI data were analyzed using the population receptive field (pRF) modeling approach. Participants also underwent ophthalmic evaluations of visual acuity and static automatic perimetry. Results: The spatial distribution and strength of pRF estimates correlated positively and significantly with clinical outcome measures in most participants with CHM. Importantly, the positive relationship between clinical and pRF measurements increased with increasing disease progression. A less consistent relationship was observed for control participants. Conclusions: Although reflecting only a small sample size, clinical evaluations of visual function in participants with CHM were well characterized by the spatial distribution and strength of pRF estimates by using a single ∼3-minute fMRI experiment. fMRI data analyzed with pRF modeling may be an efficient and objective outcome measure to complement current ophthalmic evaluations. Specifically, pRF modeling may be a feasible approach for evaluating the impact of interventions to restore visual function.

RDH12 Mutations Cause a Severe Retinal Degeneration With Relatively Spared Rod Function.

Purpose: To describe the retinal phenotype of pediatric patients with mutations in the retinol dehydrogenase 12 (RDH12) gene. Methods: Twenty-one patients from 14 families (ages 2-17 years) with RDH12-associated inherited retinal degeneration (RDH12-IRD) underwent a complete ophthalmic exam and imaging with spectral domain optical coherence tomography (SD-OCT) and near infrared and short-wavelength fundus autofluorescence. Visual field extent was measured with Goldmann kinetic perimetry, visual thresholds with dark-adapted static perimetry or with dark-adapted chromatic full-field stimulus testing (FST) and transient pupillometry. Results: Visual acuity ranged from 20/40 to light perception. There was parafoveal depigmentation or atrophic maculopathies accompanied by midperipheral intraretinal pigment migration. SD-OCT revealed foveal thinning in all patients and detectable but thinned outer nuclear layer (ONL) at greater eccentricities from the fovea. Photoreceptor outer segment (POS) signals were only detectable in small pockets within the central retina. Measurable kinetic visual fields were limited to small (<5-10°) central islands of vision. Electroretinograms were reported as undetectable or severely reduced in amplitude. FST sensitivities to a 467 nm stimulus were rod-mediated and reduced on average by ∼2.5 log units. A thinned central ONL colocalized with severely reduced to nondetectable cone-mediated sensitivities. Pupillometry confirmed the psychophysically measured abnormalities. Conclusions: RDH12-IRD causes an early-onset, retina-wide disease with particularly severe central retinal abnormalities associated with relatively less severe rod photoreceptor dysfunction, a pattern consistent with an early-onset cone-rod dystrophy. Severely abnormal POS but detectable ONL in the pericentral and peripapillary retina suggest these regions may become targets for gene therapy.

Training retinal imagers for retinopathy of prematurity (ROP) screening.

PURPOSE: To report the training/certification process of nonphysician imagers, image quality, and factors that affected image quality in the National Eye Institute sponsored multicentered e-ROP study. METHODS: Nonphysician imagers underwent rigorous training and certification in obtaining retinal images, with attention to clarity, focus, and optic disk placement. Image readers measured pupil size in pupil image and graded posterior pole, temporal, nasal, superior, and inferior retinal images and classified them as good, adequate, poor, or missing. Good and adequate images were deemed acceptable. RESULTS: In 4,003 image sessions of 1,257 infants, 3,453 (86.8%) were complete. Of 39,550 retinal images, 91.7% had acceptable quality, 5.6% poor, and 2.7% were missing. Inadequate pupil dilation negatively affected acceptable image quality: 54% acceptable images for pupil <5 mm versus 93% for >6 mm (P < 0.0001). When ventilatory equipment obstructed access to imaged infant, the percent of acceptable image quality decreased: 94% for no support versus 66.6% for oscillatory ventilation (P < 0.0001). Acceptable image quality rates improved from 87% to 90% (P = 0.03) from first 6 months to last 6 months at low patient volume centers, while high patient volume centers remained stable at 95%. CONCLUSIONS: Nonphysicians successfully obtained acceptable quality images for ROP evaluation. Skills improved with experience. Image quality was negatively affected by inadequate pupil dilation and the presence of obstructive ventilatory equipment.

Late recurrence of retinopathy of prematurity after treatment with both intravitreal bevacizumab and laser.

An infant of 36 weeks' postmenstrual age (PMA) and 25 weeks' gestation received bilateral intravitreal bevacizumab injections for type 1 retinopathy of prematurity. He underwent laser photocoagulation in both eyes 5 days later, confluent except for 1 clock hour obscured by hemorrhage in the left eye. Despite initial regression, neovascularization in both vascularized and lasered retina with plus disease recurred, requiring repeat laser bilaterally at 51 weeks' PMA and vitrectomy in the left eye at 54 weeks' PMA. Whereas late recurrence is thought to occur rarely after laser treatment, infants who have received both bevacizumab injections and laser may still require long-term surveillance for recurrence. In this case, fundus photography proved valuable for appreciating recurrent plus disease because the initial treatments had resulted in marked retinal vessel attenuation.

Assessment of signs of anterior blepharitis using standardized color photographs.

PURPOSE: To describe a standardized technique for acquiring and viewing photographic images of eyelids, assess the reproducibility and validity of a grading protocol for signs of anterior blepharitis, and to explore whether the signs depend on the eyelid or the area of the eyelid assessed. METHODS: Subjects with anterior blepharitis ranging from none to severe were examined by ophthalmologists at clinical sites. Digital images of the eyelids of subjects were acquired using a protocol that allowed for the calibration of color and luminance. Three ophthalmologists at a centralized reading center applied a novel protocol for grading features of anterior blepharitis from the digital images viewed on color-calibrated monitors. The agreement among graders was assessed using percent agreement and weighted kappa statistics (Kw), and the correlation of photographic and clinical gradings was assessed using Spearman correlation coefficients. RESULTS: Agreement among graders was excellent (Kw > 0.80) on the number of eyelid margin vessels and was substantial (Kw between 0.61 and 0.80) for erythema, collarettes, number of engorged vessels, and number of lashes. Grading of the photographic images and the clinical assessments of erythema and lid debris were moderately correlated (r = 0.27-0.45). The grades for different features depended on whether the upper or lower eyelid, eyelid skin or lid margin, and central or lateral lid were assessed. CONCLUSIONS: The application of a protocol to obtain and display calibrated digital images of eyelids supports the standardized assessment of anterior blepharitis in clinical care and research studies.

Interactive retinal vessel extraction by integrating vessel tracing and graph search.

Despite recent advances, automatic blood vessel extraction from low quality retina images remains difficult. We propose an interactive approach that enables a user to efficiently obtain near perfect vessel segmentation with a few mouse clicks. Given two seed points, the approach seeks an optimal path between them by minimizing a cost function. In contrast to the Live-Vessel approach, the graph in our approach is based on the curve fragments generated with vessel tracing instead of individual pixels. This enables our approach to overcome the shortcut problem in extracting tortuous vessels and the problem of vessel interference in extracting neighboring vessels in minimal-cost path techniques, resulting in less user interaction for extracting thin and tortuous vessels from low contrast images. It also makes the approach much faster.

Utility of digital stereo images for optic disc evaluation.

PURPOSE: To assess the suitability of digital stereo images for optic disc evaluations in glaucoma. METHODS: Stereo color optic disc images in both digital and 35-mm slide film formats were acquired contemporaneously from 29 subjects with various cup-to-disc ratios (range, 0.26-0.76; median, 0.475). Using a grading scale designed to assess image quality, the ease of visualizing optic disc features important for glaucoma diagnosis, and the comparative diameters of the optic disc cup, experienced observers separately compared the primary digital stereo images to each subject's 35-mm slides, to scanned images of the same 35-mm slides, and to grayscale conversions of the digital images. Statistical analysis accounted for multiple gradings and comparisons and also assessed image formats under monoscopic viewing. RESULTS: Overall, the quality of primary digital color images was judged superior to that of 35-mm slides (P < 0.001), including improved stereo (P < 0.001), but the primary digital color images were mostly equivalent to the scanned digitized images of the same slides. Color seemingly added little to grayscale optic disc images, except that peripapillary atrophy was best seen in color (P < 0.0001); both the nerve fiber layer (P < 0.0001) and the paths of blood vessels on the optic disc (P < 0.0001) were best seen in grayscale. The preference for digital over film images was maintained under monoscopic viewing conditions. CONCLUSIONS: Digital stereo optic disc images are useful for evaluating the optic disc in glaucoma and allow the application of advanced image processing applications. Grayscale images, by providing luminance distinct from color, may be informative for assessing certain features.