Wei Chieh Huang

Human CRB1-associated retinal degeneration: comparison with the rd8 Crb1-mutant mouse model.

PURPOSE To investigate the human disease due to CRB1 mutations and compare results with the Crb1-mutant rd8 mouse. METHODS Twenty-two patients with CRB1 mutations were studied. Function was assessed with perimetry and electroretinography (ERG) and retinal structure with optical coherence tomography (OCT). Cortical structure and function were quantified with magnetic resonance imaging (MRI). Rd8 mice underwent ERG, OCT, and retinal histopathology. RESULTS Visual acuities ranged from 20/25 to light perception. Rod ERGs were not detectable; small cone signals were recordable. By perimetry, small central visual islands were separated by midperipheral scotomas from far temporal peripheral islands. The central islands were cone mediated, whereas the peripheral islands retained some rod function. With OCT, there were small foveal islands of thinned outer nuclear layer (ONL) surrounded by thick delaminated retina with intraretinal hyperreflective lesions. MRI showed structurally normal optic nerves and only subtle changes to occipital lobe white and gray matter. Functional MRI indicated that whole-brain responses from patients were of reduced amplitude and spatial extent compared with those of normal controls. Rd8 mice had essentially normal ERGs; OCT and histopathology showed patchy retinal disorganization with pseudorosettes more pronounced in ventral than in dorsal retina. Photoreceptor degeneration was associated with dysplastic regions. CONCLUSIONS CRB1 mutations lead to early-onset severe loss of vision with thickened, disorganized, nonseeing retina. Impaired peripheral vision can persist in late disease stages. Rd8 mice also have a disorganized retina, but there is sufficient photoreceptor integrity to produce largely normal retinal function. Differences between human and mouse diseases will complicate proof-of-concept studies intended to advance treatment initiatives.

AAV-Mediated Gene Therapy in the Guanylate Cyclase (RetGC1/RetGC2) Double Knockout Mouse Model of Leber Congenital Amaurosis

Mutations in GUCY2D are associated with recessive Leber congenital amaurosis-1 (LCA1). GUCY2D encodes photoreceptor-specific, retinal guanylate cyclase-1 (RetGC1). Reports of retinal degeneration in LCA1 are conflicting; some describe no obvious degeneration and others report loss of both rods and cones. Proof of concept studies in models representing the spectrum of phenotypes is warranted. We have previously demonstrated adeno-associated virus (AAV)-mediated RetGC1 is therapeutic in GC1ko mice, a model exhibiting loss of cones only. The purpose of this study was to characterize AAV-mediated gene therapy in the RetGC1/RetGC2 double knockout (GCdko) mouse, a model lacking rod and cone function and exhibiting progressive loss of both photoreceptor subclasses. Use of this model also allowed for the evaluation of the functional efficiency of transgenic RetGC1 isozyme. Subretinal delivery of AAV8(Y733F) vector containing the human rhodopsin kinase (hGRK1) promoter driving murine Gucy2e was performed in GCdko mice at various postnatal time points. Treatment resulted in restoration of rod and cone function at all treatment ages and preservation of retinal structure in GCdko mice treated as late as 7 weeks of age. Functional gains and structural preservation were stable for at least 1 year. Treatment also conferred cortical- and subcortical-based visually-guided behavior. Functional efficiency of transgenic RetGC1 was indistinguishable from that of endogenous isozyme in congenic wild-type (WT) mice. This study clearly demonstrates AAV-mediated RetGC1 expression restores function to and preserves structure of rod and cone photoreceptors in a degenerative model of retinal guanylate cyclase deficiency, further supporting development of an AAV-based vector for treatment of LCA1.

Abnormal thickening as well as thinning of the photoreceptor layer in intermediate age-related macular degeneration.

PURPOSE To investigate the relationship between photoreceptor layers overlying and adjacent to large drusen in intermediate nonneovascular AMD. METHODS Patients with AMD (n = 41; aged 53-83 years) and elderly control subjects without eye disease (n = 10; aged 51-79 years) were studied with spectral-domain optical coherence tomography. Characteristics of large drusen (≥125 μm) were measured and the thickness of photoreceptor laminae overlying drusen and in retinal regions neighboring the drusen were quantified. RESULTS There were 750 large drusen in 63 intermediate AMD eyes studied. The width of the drusen sampled averaged 352 μm (SD = 153) and the height averaged 78 μm (SD = 31). There was significant reduction of the photoreceptor outer nuclear layer (ONL) thickness overlying 92% of the drusen. The thickness of the layer corresponding to photoreceptor inner and outer segments above drusen was also reduced, and the reduction was proportional to ONL thickness. In a substantial fraction (~20%) of normally laminated paradrusen locations sampled within ~300 μm of peak drusen height, ONL thickness was significantly increased compared with age and retinal location-matched normal values. Topographical analyses of the macula showed ONL thickening occurring in paradrusen regions as well as retinal locations distant from drusen. CONCLUSIONS Reductions in the photoreceptor laminae overlying drusen were detectable and this is consistent with histological studies revealing neuronal degeneration in AMD. ONL thickening in some macular areas of AMD eyes has not been previously reported and may be an early phenotypic marker for photoreceptor stress, as it has been speculated to be in hereditary retinal degenerations.

RPGR-associated retinal degeneration in human X-linked RP and a murine model

PURPOSE We investigated the retinal disease due to mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene in human patients and in an Rpgr conditional knockout (cko) mouse model. METHODS XLRP patients with RPGR-ORF15 mutations (n = 35, ages at first visit 5-72 years) had clinical examinations, and rod and cone perimetry. Rpgr-cko mice, in which the proximal promoter and first exon were deleted ubiquitously, were back-crossed onto a BALB/c background, and studied with optical coherence tomography and electroretinography (ERG). Retinal histopathology was performed on a subset. RESULTS Different patterns of rod and cone dysfunction were present in patients. Frequently, there were midperipheral losses with residual rod and cone function in central and peripheral retina. Longitudinal data indicated that central rod loss preceded peripheral rod losses. Central cone-only vision with no peripheral function was a late stage. Less commonly, patients had central rod and cone dysfunction, but preserved, albeit abnormal, midperipheral rod and cone vision. Rpgr-cko mice had progressive retinal degeneration detectable in the first months of life. ERGs indicated relatively equal rod and cone disease. At late stages, there was greater inferior versus superior retinal degeneration. CONCLUSIONS RPGR mutations lead to progressive loss of rod and cone vision, but show different patterns of residual photoreceptor disease expression. Knowledge of the patterns should guide treatment strategies. Rpgr-cko mice had onset of degeneration at relatively young ages and progressive photoreceptor disease. The natural history in this model will permit preclinical proof-of-concept studies to be designed and such studies should advance progress toward human therapy.

Inner and Outer Retinal Changes in Retinal Degenerations Associated With ABCA4 Mutations

PURPOSE To investigate in vivo inner and outer retinal microstructure and effects of structural abnormalities on visual function in patients with retinal degeneration caused by ABCA4 mutations (ABCA4-RD). METHODS Patients with ABCA4-RD (n = 45; age range, 9-71 years) were studied by spectral-domain optical coherence tomography (OCT) scans extending from the fovea to 30° eccentricity along horizontal and vertical meridians. Thicknesses of outer and inner retinal laminae were analyzed. Serial OCT measurements available over a mean period of 4 years (range, 2-8 years) allowed examination of the progression of outer and inner retinal changes. A subset of patients had dark-adapted chromatic static threshold perimetry. RESULTS There was a spectrum of photoreceptor layer thickness changes from localized central retinal abnormalities to extensive thinning across central and near midperipheral retina. The inner retina also showed changes. There was thickening of the inner nuclear layer (INL) that was mainly associated with regions of photoreceptor loss. Serial data documented only limited change in some patients while others showed an increase in outer nuclear layer (ONL) thinning accompanied by increased INL thickening in some regions imaged. Visual function in regions both with and without INL thickening was describable with a previously defined model based on photoreceptor quantum catch. CONCLUSIONS Inner retinal laminar abnormalities, as in other human photoreceptor diseases, can be a feature of ABCA4-RD. These changes are likely due to the retinal remodeling that accompanies photoreceptor loss. Rod photoreceptor-mediated visual loss in retinal regionswith inner laminopathy at the stages studied did not exceed the prediction from photoreceptor loss alone.

Gene Therapy for Retinitis Pigmentosa Caused by MFRP Mutations: Human Phenotype and Preliminary Proof of Concept

Autosomal recessive retinitis pigmentosa (RP), a heterogeneous group of degenerations of the retina, can be due to mutations in the MFRP (membrane-type frizzled-related protein) gene. A patient with RP with MFRP mutations, one of which is novel and the first splice site mutation reported, was characterized by noninvasive retinal and visual studies. The phenotype, albeit complex, suggested that this retinal degeneration may be a candidate for gene-based therapy. Proof-of-concept studies were performed in the rd6 Mfrp mutant mouse model. The fast-acting tyrosine-capsid mutant AAV8 (Y733F) vector containing the small chicken β-actin promoter driving the wild-type mouse Mfrp gene was used. Subretinal vector delivery on postnatal day 14 prevented retinal degeneration. Treatment rescued rod and cone photoreceptors, as assessed by electroretinography and retinal histology at 2 months of age. This AAV-mediated gene delivery also resulted in robust MFRP expression predominantly in its normal location within the retinal pigment epithelium apical membrane and its microvilli. The clinical features of MFRP-RP and our preliminary data indicating a response to gene therapy in the rd6 mouse suggest that this form of RP is a potential target for gene-based therapy.

TULP1 mutations causing early-onset retinal degeneration: preserved but insensitive macular cones.

PURPOSE To investigate visual function and outer and inner retinal structure in the rare form of retinal degeneration (RD) caused by TULP1 (tubby-like protein 1) mutations. METHODS Retinal degeneration patients with TULP1 mutations (n = 5; age range, 5-36 years) were studied by kinetic and chromatic static perimetry, en face autofluorescence imaging, and spectral-domain optical coherence tomography (OCT) scans. Outer and inner retinal laminar thickness were measured and mapped across the central retina. Comparisons were made with results from patients with RD associated with four ciliopathy genotypes (MAK, RPGR, BBS1, and USH2A). RESULTS The TULP1-RD patients were severely affected already in the first decade of life and there was rapidly progressive visual loss. No evidence of rod function was present at any age. Small central islands showed melanized retinal pigment epithelium by autofluorescence imaging and well-preserved photoreceptor laminar thickness by OCT imaging. There was extracentral loss of laminar architecture and increased inner retinal thickening. Structure-function relationships in residual foveal cone islands were made in TULP1-RD patients and in other retinopathies considered ciliopathies. Patients with TULP1-RD, unlike the others, had greater dysfunction for the degree of foveal structural preservation. CONCLUSIONS Retinal degeneration with TULP1 mutations leads to a small central island of residual foveal cones at early ages. These cones are less sensitive than expected from the residual structure. The human phenotype is consistent with experimental evidence in the Tulp1 knockout mouse model that visual dysfunction could be complicated by abnormal processes proximal to cone outer segments.

Molecular Heterogeneity Within the Clinical Diagnosis of Pericentral Retinal Degeneration

PURPOSE To characterize in detail the phenotype and genotype of patients with pericentral retinal degeneration (PRD). METHODS Patients were screened for an annular ring scotoma ranging from 3° to 40° (n = 28, ages 24-71) with kinetic perimetry. All patients had pigmentary retinopathy in the region of the dysfunction. Further studies included cross-sectional and en face imaging, static chromatic perimetry, and electroretinography. Molecular screening was performed. RESULTS Genotypes of 14 of 28 PRD patients were identified: There were mutations in eight different genes previously associated with autosomal dominant or autosomal recessive RDs. Kinetic fields monitored in some patients over years to more than a decade could be stable or show increased extent of the scotoma. Electroretinograms were recordable but with different severities of dysfunction. Patterns of photoreceptor outer nuclear layer (ONL) loss corresponded to the distribution of visual dysfunction. Outer nuclear layer thickness topography and en face imaging indicated that the greatest disease expression was in the area of known highest rod photoreceptor density. CONCLUSIONS Molecular heterogeneity was a feature of the PRD phenotype. Many of the molecular causes were also associated with other phenotypes, such as maculopathies, typical retinitis pigmentosa (RP) and cone-rod dystrophy. The pericentral pattern of retinal degeneration is thus confirmed to be an uncommon phenotype of many different genotypes rather than a distinct disease entity.

Leber Congenital Amaurosis: Genotypes and Retinal Structure Phenotypes.

Leber congenital amaurosis (LCA) patients of 10 known genotypes (n = 24; age range, 3-25 years) were studied clinically and by optical coherence tomography (OCT). Comparisons were made between OCT results across the horizontal meridian (central 60(o)) of the patients. Three patterns were identified. First, there were LCA genotypes with unusual and readily identifiable patterns, such as near normal outer nuclear layer (ONL) across the central retina or severely dysplastic retina. Second, there were genotypes with well-formed foveal architecture but only residual central islands of normal or reduced ONL thickness. Third, some genotypes showed central ONL losses or dysmorphology suggesting early macular disease or foveal maldevelopment. Objective in vivo morphological features could complement other phenotypic characteristics and help guide genetic testing of LCA patients or at least permit a differential diagnosis of genotypes to be made in the clinic.