Ioannis S. Dimopoulos

Pathogenic mechanisms and the prospect of gene therapy for choroideremia.

Introduction: Choroideremia is a rare, X-linked disorder recognized by its specific ocular phenotype as a progressive degenerative retinopathy resulting in blindness. New therapeutic approaches, primarily based on genetic mechanisms, have emerged that aim to prevent the progressive vision loss. Areas covered: This article will review the research that has progressed incrementally over the past two decades from mapping to gene discovery, uncovering the presumed mechanisms triggering the retinopathy to preclinical testing of potential therapies. Expert opinion: While still in an evaluative phase, the introduction of gene replacement as a potential therapy has been greeted with great enthusiasm by patients, advocacy groups and the medical community.

Microperimetry as an Outcome Measure in Choroideremia Trials: Reproducibility and Beyond.

PURPOSE To determine test-retest repeatability of microperimetry testing (MP) in choroideremia (CHM) subjects using standard and personalized stimulus grids. METHODS Fifteen CHM subjects (28 eyes) underwent consecutive repeat examinations with the Macular Integrity Assessment (MAIA) microperimeter using a standard (10°) and a customized macular grid adapted to individual macular pathology. Repeatability of standard-grid mean (MS) and point-wise (PWS) sensitivity was determined and compared with age-matched controls (seven eyes), with PWS separately analyzed for loci within and outside the border of degeneration. Interpolated volumetric indices were used to estimate repeatability of customized grids and compare their performance to standard grids. RESULTS Test-retest measures of standard-grid MS yielded higher coefficients of variation (CV) in CHM subjects compared with controls (0.09 vs. 0.02). Volumetric indices from customized grids improved repeatability by driving CV values to 0.05 and close to 0.02 for region-of-interest (ROI) analysis. Variability of PWS was significantly higher in CHM, especially at the border of degeneration (10.68 vs. 4.74 dB at the central retina, P < 0.001). CONCLUSIONS Microperimetry testing in CHM shows high test-retest variation at the border of degeneration, which influences repeatability of MS measures. Volumetric measures from customized grids can improve reliability of both global and regional sensitivity assessment. Nevertheless, inherent test-retest variation of individual points needs to be taken into account when assessing potential functional decline and/or disease progression.

Early Onset Ultrastructural and Functional Defects in RPE and Photoreceptors of a Stargardt-Like Macular Dystrophy (STGD3) Transgenic Mouse Model

PURPOSE We investigated the interplay between photoreceptors expressing mutant ELOVL4 (responsible for Stargardt-like disease, STGD3) and RPE in the initial stages of retinal degeneration. METHODS Using electron microscopy and electroretinogram (ERG), we assessed RPE and photoreceptor ultrastructure and function in transgenic ELOVL4 (TG1-2 line; TG) and wild-type (WT) littermates. Experiments were done at P30, 1 month before photoreceptor loss in TG and at P90, a time point with approximately 30% rod loss. To further elucidate the mechanism underlying our ultrastructural and functional results, we undertook Western blotting and immunohistochemistry of key proteins involved in phagocytosis of outer segments by RPE cells. RESULTS Firstly, we showed that in TG mouse photoreceptors, endogenous ELOVL4 protein is not mislocalized in the presence of the mutated ELOVL4 protein. Secondly, we found evidence of RPE toxicity at P30, preceding any photoreceptor loss. Pathology in RPE cells was exacerbated at P90. Furthermore, higher proportions of phagosomes remained at the apical side of RPE cells. Subretinal lysosomal deposits were immunopositive for phagocytic proteins. Ultrastructural analysis of photoreceptor (rod) outer segments showed disrupted surface morphology consisting of disc spacing irregularities. Finally, rods and RPE exhibited signs of dysfunction as measured by the ERG a-wave leading edge (P30) and c-wave (P90), respectively. CONCLUSIONS The presence of human mutant ELOVL4 in transgenic mouse photoreceptors leads to early outer segment disc pathology and RPE cytotoxicity. Defective processing of these abnormal discs by RPE cells ultimately may be responsible for outer segment truncation, photoreceptor death, and vision loss.

Choroideremia research: Report and perspectives on the second international scientific symposium for choroideremia

ABSTRACT Purpose: To discuss progress in research on choroideremia (CHM) and related retinopathies with special emphasis on gene therapy approaches. Methods: Biomedical and clinical researchers from across the world as well as representatives of the social science research community were convened to the 2nd International Scientific Symposium for Choroideremia in Denver, Colorado in June 2014 to enhance our understanding of CHM and accelerate the translation of research to clinical application for the benefit of those affected by CHM. Results: Pre-clinical research using cell and animal models continues to further our understanding in the pathogenesis of CHM as well as to demonstrate proof-of-concept for gene transfer strategies. With the advent of modern imaging technology, better outcome measures are being defined for upcoming clinical trials. Results from the first gene therapy trial in CHM show promise, with sustained visual improvement over 6 months post-treatment. Current and next-generation gene transfer approaches may make targeted vector delivery possible in the future for CHM and other inherited retinal diseases. Conclusions: While no accepted therapies exist for CHM, promising approaches using viral-vectored gene therapy and cell therapies are entering clinical trials for eye diseases, with gene therapy trials underway for CHM.

Changes in rod and cone-driven oscillatory potentials in the aging human retina.

PURPOSE We recorded oscillatory potentials (OPs) to document how age impacts on rod- and cone-driven inner retina function. METHODS Dark- and light-adapted electroretinogram (ERG) luminance-response functions were recorded in healthy human subjects aged 20 to 39, 40 to 59, and 60 to 82 years. Raw ERG traces (0.1-300 Hz) were filtered (75-300 Hz) to measure OPs trough-to-peak in the time-amplitude domain. Morlet wavelet transform (MWT) allowed documenting OPs time-amplitude-frequency distribution from raw traces. RESULTS Under dark adaptation, both methods revealed reduced OP amplitudes and prolonged implicit times by 40 years of age. The MWT identified a high-frequency band as the main oscillator, which frequency (150-155 Hz) was unaffected by age. Under light adaptation, most OP peaks were delayed by 40 years of age. Peak-trough measures yielded inconsistent results in relation to luminance. Contrastingly, MWT distinguished two frequency bands at all luminances: high frequency (135 ± 6 Hz) time locked to the onset of early OPs and low frequency (82 ± 7 Hz), giving rise to early and late OPs. By 60 years, there was a consistent power reduction specific to the low-frequency band. CONCLUSIONS Age-related OP changes precede those seen with a- (photoreceptoral) and b-waves (postphotoreceptoral). In addition, MWT allows quantifying distinct low- and high-frequency oscillators in the human retina, which complement traditional OP analysis methods. The identification of an age-independent OP marker (light-adapted high frequency band) opens a new dimension for the screening of retinal degenerations and their impact on inner retina function.

Subjects With Unilateral Neovascular AMD Have Bilateral Delays in Rod-Mediated Phototransduction Activation Kinetics and in Dark Adaptation Recovery

PURPOSE To assess the impact of early (dry) and late (wet/neovascular and/or atrophic) forms of AMD on panretinal function. METHODS Light- and dark-adapted full-field ERG recordings were obtained over a 5-log-unit intensity range from both eyes of 25 patients with unilateral wet AMD. Fellow eyes showed various signs of dry AMD ranging from multiple medium-sized drusen to noncentral geographic atrophy. The leading edges of rod-isolated ERG a-waves were fitted to a quantitative model of phototransduction. ERG a- and b-wave amplitudes and implicit times were compared between wet and dry AMD eyes and from non-AMD eyes of age-matched subjects. A quantitative and objective assessment of dark adaptation was achieved by recording the recovery of the pure rod b-wave (postsynaptic depolarization of rod bipolar cells); b-wave amplitudes were measured at 120-second intervals for 20 minutes and normalized to the amplitude recorded at t = 20 minutes. RESULTS Delays in mixed a- and b-wave implicit times were recorded in both wet and dry AMD eyes. Time required to reach 50% of fully recovered responses was delayed in all wet and dry AMD eyes independently of dry AMD severity in the fellow eye. Generalized cone dysfunction and slower activation of the rod phototransduction cascade was noted in a subgroup of patients with advanced features of dry AMD in the fellow eye. CONCLUSIONS Patients with unilateral wet AMD display rod dysfunction in both their wet and dry AMD eyes. A subset of these patients display, in addition, bilateral cone dysfunction and delayed rod phototransduction activation, which may either reflect extensive morphologic change in advanced stages of AMD and/or represent a distinct phenotypic manifestation within the heterogeneous context of AMD as a disease.