Safety and improved efficacy signals following gene therapy in childhood blindness caused by GUCY2D mutations

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

Expression of the Streptomyces coelicolor SoxR Regulon Is Intimately Linked with Actinorhodin Production▿ †

The [2Fe-2S]-containing transcription factor SoxR is conserved in diverse bacteria. SoxR is traditionally known as the regulator of a global oxidative stress response in Escherichia coli, but recent studies suggest that this function may be restricted to enteric bacteria. In the vast majority of nonenterics, SoxR is predicted to mediate a response to endogenously produced redox-active metabolites. We have examined the regulation and function of the SoxR regulon in the model antibiotic-producing filamentous bacterium Streptomyces coelicolor. Unlike the E. coli soxR deletion mutant, the S. coelicolor equivalent is not hypersensitive to oxidants, indicating that SoxR does not potentiate antioxidant defense in the latter. SoxR regulates five genes in S. coelicolor, including those encoding a putative ABC transporter, two oxidoreductases, a monooxygenase, and a possible NAD-dependent epimerase/dehydratase. Expression of these genes depends on the production of the benzochromanequinone antibiotic actinorhodin and requires intact [2Fe-2S] clusters in SoxR. These data indicate that actinorhodin, or a redox-active precursor, modulates SoxR activity in S. coelicolor to stimulate the production of a membrane transporter and proteins with homology to actinorhodin-tailoring enzymes. While the role of SoxR in S. coelicolor remains under investigation, these studies support the notion that SoxR has been adapted to perform distinct physiological functions to serve the needs of organisms that occupy different ecological niches and face different environmental challenges

Leber Congenital Amaurosis Due to <i>GUCY2D</i> 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

Drusen and Photoreceptor Abnormalities in African-Americans with Intermediate Non-neovascular Age-related Macular Degeneration

Purpose/Aim: To investigate the relationship of drusen and photoreceptor abnormalities in African-American (AA) patients with intermediate non-neovascular age-related macular degeneration (AMD). Materials and methods: AA patients with intermediate AMD (n = 11; age 52-77 years) were studied with spectral-domain optical coherence tomography. Macular location and characteristics of large drusen (≥125 µm) were determined. Thickness of photoreceptor laminae was quantified overlying drusen and in other macular regions. A patient with advanced AMD (age 87) was included to illustrate the disease spectrum. Results: In this AA patient cohort, the spectrum of changes known to occur in AMD, including large drusen, sub-retinal drusenoid deposits and geographic atrophy, were identified. In intermediate AMD eyes (n = 17), there were 183 large drusen, the majority of which were pericentral in location. Overlying the drusen there was significant thinning of the photoreceptor outer nuclear layer (termed ONL + ) as well as the inner and outer segments (IS + OS). The reductions in IS + OS thickness were directly related to ONL + thickness. In a fraction (∼8%) of paradrusen locations with normal lamination sampled within ∼280 µm of peak drusen height, ONL + was significantly thickened compared to age and retinal-location-matched normal values. Topographical maps of the macula confirmed ONL thickening in regions neighboring and distant to large drusen. Conclusions: We confirm there is a pericentral distribution of drusen across AA-AMD maculae rather than the central localization in Caucasian AMD. Reductions in the photoreceptor laminae overlying drusen are evident. ONL + thickening in some macular areas of AA-AMD eyes may be an early phenotypic marker for photoreceptor stress

Short-Wavelength Sensitive Cone (S-cone) Testing as an Outcome Measure for <i>NR2E3</i> Clinical Treatment Trials

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

Blue cone monochromacy: visual function and efficacy outcome measures for clinical trials.

BackgroundBlue Cone Monochromacy (BCM) is an X-linked retinopathy caused by mutations in the OPN1LW / OPN1MW gene cluster, encoding long (L)- and middle (M)-wavelength sensitive cone opsins. Recent evidence shows sufficient structural integrity of cone photoreceptors in BCM to warrant consideration of a gene therapy approach to the disease. In the present study, the vision in BCM is examined, specifically seeking clinically-feasible outcomes for a future clinical trial.MethodsBCM patients (n = 25, ages 5-72) were studied with kinetic and static chromatic perimetry, full-field sensitivity testing, and eye movement recordings. Vision at the fovea and parafovea was probed with chromatic microperimetry.ResultsKinetic fields with a Goldmann size V target were generally full. Short-wavelength (S-) sensitive cone function was normal or near normal in most patients. Light-adapted perimetry results on conventional background lights were abnormally reduced; 600-nm stimuli were seen by rods whereas white stimuli were seen by both rods and S-cones. Under dark-adapted conditions, 500-nm stimuli were seen by rods in both BCM and normals. Spectral sensitivity functions in the superior retina showed retained rod and S-cone functions in BCM under dark-adapted and light-adapted conditions. In the fovea, normal subjects showed L/M-cone mediation using a 650-nm stimulus under dark-adapted conditions, whereas BCM patients had reduced sensitivity driven by rod vision. Full-field red stimuli on bright blue backgrounds were seen by L/M-cones in normal subjects whereas BCM patients had abnormally reduced and rod-mediated sensitivities. Fixation location could vary from fovea to parafovea. Chromatic microperimetry demonstrated a large loss of sensitivity to red stimuli presented on a cyan adapting background at the anatomical fovea and surrounding parafovea.ConclusionsBCM rods continue to signal vision under conditions normally associated with daylight vision. Localized and retina-wide outcome measures were examined to evaluate possible improvement of L/M-cone-based vision in a clinical trial

Visual fields of BCM patients evaluated with kinetic and static perimetry.

<p>(A) Light-adapted (LA) vertical sensitivity profiles from a normal subject and a BCM patient using achromatic (black line) and 600-nm (orange line) stimuli on a 10 cd.m^-2 white background, or 440-nm (blue line) stimuli on a yellow background (YB). (B) S-cone sensitivity profiles (filled circles) of the BCM patients using a 440-nm stimulus on YB compared to normal limits (gray = ±2SD). (C) LA white vertical sensitivity profiles of BCM patients (filled circles) compared to normal (gray). Blue line is the S-cone sensitivities from Panel C shifted according to the difference in effectiveness between the white and 440-nm stimuli. (D) LA 600-nm vertical sensitivity profiles of BCM patients (filled circles) compared to normal (gray). (E) Sensitivity differences between LA white and LA 600-nm stimuli are shown for the BCM patients (filled circles) and normal (unfilled circles). Predicted differences for rod (green dashes) and L/M cone (orange dashes) mediation are shown. (F) Dark-adapted (DA) vertical sensitivity profiles from a normal subject and a BCM patient using 500-nm (green line) and 650-nm (red line) stimuli. Above the results it is shown whether there is rod (R) or mixed (M) mediation, as determined by the differences between sensitivities to the stimuli. (G) DA 500-nm vertical sensitivity profiles of BCM patients (filled circles) compared to normal (gray). (H) DA 650 nm vertical sensitivity profiles of BCM patients (filled circles) compared to normal (gray). (I) Sensitivity differences between DA 500- and DA 650-nm stimuli are consistent with rod mediation (gray) at all locations except for the normal results with 650 nm at fixation. S, superior; I, inferior. (J) DA 650-nm sensitivities at fixation in normal and BCM. Normal 650-nm sensitivities are mediated by the L/M cones (C) whereas BCM sensitivities are mediated by the rods (R). Error bars are ±1SD.</p

Nystagmus and foveal function in BCM.

<p>(A) Fixation locations in a normal subject and 3 BCM patients. For each subject, 10 s long epochs of eye movement data during fixation to a large visible red target (Target I) are shown in spatial (left) and spatio-temporal (right) coordinates. Spatial distribution of fixation clouds are shown on infrared SLO images of each macula with standard circles centered on the anatomical foveal depression. Spatio-temporal distribution of eye movements are shown on chart records for X and Y directions; up is nasal retina for X and superior retina for Y. All results are presented as equivalent right eyes for comparability. Horizontal dashed lines on the chart records depict the location of the anatomical fovea. (B) Fixation location and instability in BCM patients as a function of the bright red standard target (I), a green target (II) scotopically-matched to the standard target but expected to show greater visibility to S-cones, and a dim red target (III). N.S., not significant; *, P<0.05. (C) Distribution of fixation locations with the standard target in all patients. I = inferior, N = nasal, S = superior, and T = temporal retina. (D) Fixation location and instability as a function of best-corrected visual acuity. (E) Test pattern used with microperimetric stimuli to evaluate visual function under chromatic adaptation displayed on a normal near-infrared reflectance image. Stimulus locations are divided into 5 groups; f, foveal region, s, superior, i, inferior, t, temporal and n, nasal retina. (F,G) Sensitivities to blue stimuli on yellow background (BonY) and red stimuli on cyan background (RonC) in individual BCM patients (bars left to right; P2, P3, P4, P6, P8, P9, P10, P15, P16, P17, P18, P20, P25, P26, P28, and P29) compared to normal results (symbols; mean ±1sd) at the five regions shown in panel E. BCM results plotted below the zero line in Panel G represent those cases where the brightest available stimulus was not seen.</p

Spectral sensitivity functions in normal subjects and BCM patients recorded at 14° superior field.

<p>(A) Sensitivities (mean±1 SD) to six spectrally distinct stimuli in normal subjects (n = 3) under dark-adapted (left), and on 1 (middle) and 10 cd.m^-2 (right) white backgrounds. (B) Sensitivities to the spectrally distinct stimuli in BCM patients for the same three adaptation conditions as in Panel A. Results from P8 are shown at the correct ordinate location; results from remaining patients have been adjusted by 1 log increments for visibility. Theoretical functions describing rod (green), S cone (blue), L/M cone (orange) sensitivities are shown after vertical shifts to fit relevant normal and BCM data in Panels A and B. (C) Comparison of individual normal and BCM sensitivities at 500 nm. (D,E) Comparison of individual normal and BCM sensitivities at 440, 500 and 560 nm. Symbols in Panels C, D, and E are painted by colors derived from the fit of theoretical functions to the spectral data. N = normal, B = BCM.</p