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1.  Human Cone Visual Pigment Deletions Spare Sufficient Photoreceptors to Warrant Gene Therapy 
Human Gene Therapy  2013;24(12):993-1006.
Abstract
Human X-linked blue-cone monochromacy (BCM), a disabling congenital visual disorder of cone photoreceptors, is a candidate disease for gene augmentation therapy. BCM is caused by either mutations in the red (OPN1LW) and green (OPN1MW) cone photoreceptor opsin gene array or large deletions encompassing portions of the gene array and upstream regulatory sequences that would predict a lack of red or green opsin expression. The fate of opsin-deficient cone cells is unknown. We know that rod opsin null mutant mice show rapid postnatal death of rod photoreceptors. Using in vivo histology with high-resolution retinal imaging, we studied a cohort of 20 BCM patients (age range 5–58) with large deletions in the red/green opsin gene array. Already in the first years of life, retinal structure was not normal: there was partial loss of photoreceptors across the central retina. Remaining cone cells had detectable outer segments that were abnormally shortened. Adaptive optics imaging confirmed the existence of inner segments at a spatial density greater than that expected for the residual blue cones. The evidence indicates that human cones in patients with deletions in the red/green opsin gene array can survive in reduced numbers with limited outer segment material, suggesting potential value of gene therapy for BCM.
doi:10.1089/hum.2013.153
PMCID: PMC3868405  PMID: 24067079
2.  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.
ABCA4-retinal degenerations were studied for inner retinal microstructural changes in patients representing a wide spectrum of outer retinopathy. Inner retinal laminar defects were associated with outer retinal abnormality. Visual dysfunction in retina with inner laminopathy was not more than expected from photoreceptor loss alone.
doi:10.1167/iovs.13-13768
PMCID: PMC3968929  PMID: 24550365
retinal remodeling; optical coherence tomography; Stargardt disease
3.  Intervisit Variability of Visual Parameters in Leber Congenital Amaurosis Caused by RPE65 Mutations 
Purpose.
To determine the intervisit variability of kinetic visual fields and visual acuity in patients with Leber congenital amaurosis (LCA) caused by mutations in the RPE65 (Retinal Pigment Epithelium–specific protein 65kDa) gene.
Methods.
RPE65-LCA patients (n = 20; ages 11–40 years) were studied on at least two visits separated by fewer than 120 days using Goldmann visual field (GVF) and ETDRS visual acuity (VA) in a retrospective review. GVFs were quantified by computing the spherical coordinates of their vertices and calculating the solid angle subtended, and reported in normalized solid-angle units (nsu) as a percentage of average normal field extent. Repeatability coefficients were calculated using 95% confidence intervals on log10-converted variables.
Results.
Visual field extents in RPE65-LCA spanned a wide range from 4 to 95 nsu. The repeatability coefficient was 0.248 (log10nsu), suggesting cutoffs for significant change (in nsu) of +77% for improvement and −44% for worsening. VA in RPE65-LCA ranged from logMAR = 0.14 to 1.96 (20/40 to 20/1250). The repeatability coefficient was 0.170 (logMAR) (±8.5 ETDRS letters). Comparisons with published studies of ungenotyped retinitis pigmentosa showed that the RPE65-LCA patients had higher variability in kinetic field extent. VA variability in RPE65-LCA fell within reported results for retinitis pigmentosa.
Conclusions.
Variability data for GVF and VA are provided to permit interpretation of the significance of increases and decreases of these functional outcomes in ongoing and planned clinical trials of therapy for LCA caused by RPE65 mutations.
RPE65-LCA was studied for intervisit variability of outcome measures currently used in clinical trials. Variability limits for visual acuity were like those in RP, but there was higher variability in kinetic visual fields, suggesting the need for a disease-specific approach.
doi:10.1167/iovs.12-11341
PMCID: PMC3597193  PMID: 23341016
4.  Gene Therapy for Retinitis Pigmentosa Caused by MFRP Mutations: Human Phenotype and Preliminary Proof of Concept 
Human Gene Therapy  2011;23(4):367-376.
Abstract
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.
In this proof-of-concept study, Dinculescu and colleagues demonstrate that subretinal delivery of a self-complementary tyrosine-capsid mutant AAV serotype 8 (AAV8) (Y733F) vector carrying the mouse Mfrp gene prevents retinal degeneration and rescues rod and cone photoreceptors in a mouse model of autosomal recessive retinitis pigmentosa.
doi:10.1089/hum.2011.169
PMCID: PMC3327606  PMID: 22142163
5.  Gene Therapy for Leber Congenital Amaurosis caused by RPE65 mutations: Safety and Efficacy in Fifteen Children and Adults Followed up to Three Years 
Archives of ophthalmology  2011;130(1):9-24.
Objective
To determine safety and efficacy of subretinal gene therapy in the RPE65 form of Leber congenital amaurosis using recombinant adeno-associated virus 2 (rAAV2) carrying human RPE65 gene.
Design
Open-label, dose-escalation Phase I study of 15 patients (11-30 years) evaluated after subretinal injection of rAAV2-hRPE65 to the worse-functioning eye. Five cohorts represented four dose levels and two different injection strategies.
Main Outcome Measures
Primary outcomes were systemic and ocular safety. Secondary outcomes assayed visual function with dark-adapted full-field sensitivity testing and ETDRS visual acuity. Further assays included immune responses to the vector, static visual fields, pupillometry, mobility performance and OCT.
Results
No systemic toxicity was detected; ocular adverse events were related to surgery. Visual function improved in all patients to different degrees; improvements were localized to treated areas. Cone and rod sensitivities increased significantly in study eyes but not control eyes. Minor acuity improvements were recorded in many study and control eyes. Major acuity improvements occurred in study eyes with the lowest entry acuities and parafoveal fixation loci treated with subretinal injections. Other patients with better foveal structure lost retinal thickness and acuity after subfoveal injections.
Conclusions
RPE65-LCA gene therapy is sufficiently safe and substantially efficacious to the extrafoveal retina. There is no benefit and some risk in treating the fovea. No evidence of age-dependent effects was found. Our results point to specific treatment strategies for subsequent phases.
Application to Clinical Practice
Gene therapy for inherited retinal disease has the potential to become a future part of clinical practice.
doi:10.1001/archophthalmol.2011.298
PMCID: PMC3600816  PMID: 21911650
6.  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.
Progress in treating canine RPGR disease prompted us to characterize patients with RPGR-ORF15 mutations and provide a detailed natural history of a novel Rpgr-mutant mouse for further proof-of-concept experiments.
doi:10.1167/iovs.12-10070
PMCID: PMC3422104  PMID: 22807293
7.  Rescue of Enzymatic Function for Disease-associated RPE65 Proteins Containing Various Missense Mutations in Non-active Sites* 
The Journal of Biological Chemistry  2014;289(27):18943-18956.
Background: Disease-causing mutations reduce RPE65 protein levels with unknown mechanisms.
Results: Interaction of 26 S proteasome non-ATPase regulatory subunit 13 with mutant RPE65s mediates degradation of misfolded RPE65s via the ubiquitin-proteasome pathway.
Conclusion: Many mutant RPE65s with non-active site mutations are catalytically active and can be rescued.
Significance: Low temperature and chaperones that rescue enzymatic function of mutant RPE65s are therapeutic candidates.
Over 70 different missense mutations, including a dominant mutation, in RPE65 retinoid isomerase are associated with distinct forms of retinal degeneration; however, the disease mechanisms for most of these mutations have not been studied. Although some mutations have been shown to abolish enzyme activity, the molecular mechanisms leading to the loss of enzymatic function and retinal degeneration remain poorly understood. Here we show that the 26 S proteasome non-ATPase regulatory subunit 13 (PSMD13), a newly identified negative regulator of RPE65, plays a critical role in regulating pathogenicity of three mutations (L22P, T101I, and L408P) by mediating rapid degradation of mutated RPE65s via a ubiquitination- and proteasome-dependent non-lysosomal pathway. These mutant RPE65s were misfolded and formed aggregates or high molecular complexes via disulfide bonds. Interaction of PSMD13 with mutant RPE65s promoted degradation of misfolded but not properly folded mutant RPE65s. Many mutations, including L22P, T101I, and L408P, were mapped on non-active sites. Although their activities were very low, these mutant RPE65s were catalytically active and could be significantly rescued at low temperature, whereas mutant RPE65s with a distinct active site mutation could not be rescued under the same conditions. Sodium 4-phenylbutyrate and glycerol displayed a significant synergistic effect on the low temperature rescue of the mutant RPE65s by promoting proper folding, reducing aggregation, and increasing membrane association. Our results suggest that a low temperature eye mask and sodium 4-phenylbutyrate, a United States Food and Drug Administration-approved oral medicine, may provide a promising “protein repair therapy” that can enhance the efficacy of gene therapy by reducing the cytotoxic effect of misfolded mutant RPE65s.
doi:10.1074/jbc.M114.552117
PMCID: PMC4081934  PMID: 24849605
Aggregation; Membrane; Photoreceptor; Proteasome; Retinal Degeneration; Retinoid; Ubiquitination; RPE65; PSMD13; Leber Congenital Amaurosis
8.  Retinal Disease Course in Usher Syndrome 1B Due to MYO7A Mutations 
The promise of clinical trials of treatment for Usher syndromes requires moving forward from the classic three clinical subtypes to some greater understanding of how the different USH diseases are expressed. Within this study's cohort of USH1B patients, there were differences in severity of rod disease and photoreceptor cell loss, and the study inquired whether the predicted consequence of the mutant MYO7A alleles could help explain the observed variations in phenotype.
Purpose.
To determine the disease course in Usher syndrome type IB (USH1B) caused by myosin 7A (MYO7A) gene mutations.
Methods.
USH1B patients (n = 33, ages 2–61) representing 25 different families were studied by ocular examination, kinetic and chromatic static perimetry, dark adaptometry, and optical coherence tomography (OCT). Consequences of the mutant alleles were predicted.
Results.
All MYO7A patients had severely abnormal ERGs, but kinetic fields revealed regional patterns of visual loss that suggested a disease sequence. Rod-mediated vision could be lost to different degrees in the first decades of life. Cone vision followed a more predictable and slower decline. Central vision ranged from normal to reduced in the first four decades of life and thereafter was severely abnormal. Dark adaptation kinetics was normal. Photoreceptor layer thickness in a wide region of central retina could differ dramatically between patients of comparable ages; and there were examples of severe losses in childhood as well as relative preservation in patients in the third decade of life. Comparisons were made between the mutant alleles in mild versus more severe phenotypes.
Conclusions.
A disease sequence in USH1B leads from generally full but impaired visual fields to residual small central islands. At most disease stages, there was preserved temporal peripheral field, a potential target for early phase clinical trials of gene therapy. From data comparing patients' rod disease in this cohort, the authors speculate that null MYO7A alleles could be associated with milder dysfunction and fewer photoreceptor structural losses at ages when other genotypes show more severe phenotypes.
doi:10.1167/iovs.11-8313
PMCID: PMC3263772  PMID: 21873662
9.  Human CRB1-Associated Retinal Degeneration: Comparison with the rd8 Crb1-Mutant Mouse Model 
The common form of Leber congenital amaurosis (LCA) due to CRB1 mutations needs further characterization of the human disease and a test of relevance of currently available animal models. A cohort of these patients was evaluated, and the disease expression was compared to that of the rd8 mouse model, to seek answers to questions of how to move CRB1-LCA closer to therapy.
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.
doi:10.1167/iovs.11-7701
PMCID: PMC3176016  PMID: 21757580
10.  Gene Therapy for Rare Diseases: Summary of a National Institutes of Health Workshop, September 13, 2012 
Human Gene Therapy  2013;24(4):355-362.
Abstract
Gene therapy has shown clinical efficacy for several rare diseases, using different approaches and vectors. The Gene Therapy for Rare Diseases workshop, sponsored by the National Institutes of Health (NIH) Office of Biotechnology Activities and Office of Rare Diseases Research, brought together investigators from different disciplines to discuss the challenges and opportunities for advancing the field including means for enhancing data sharing for preclinical and clinical studies, development and utilization of available NIH resources, and interactions with the U.S. Food and Drug Administration.
doi:10.1089/hum.2013.064
PMCID: PMC3631014  PMID: 23517518
11.  Natural History of Cone Disease in the Murine Model of Leber Congenital Amaurosis Due to CEP290 Mutation: Determining the Timing and Expectation of Therapy 
PLoS ONE  2014;9(3):e92928.
Background
Mutations in the CEP290 (cilia-centrosomal protein 290 kDa) gene in Leber congenital amaurosis (LCA) cause early onset visual loss but retained cone photoreceptors in the fovea, which is the potential therapeutic target. A cone-only mouse model carrying a Cep290 gene mutation, rd16;Nrl−/−, was engineered to mimic the human disease. In the current study, we determined the natural history of retinal structure and function in this murine model to permit design of pre-clinical proof-of-concept studies and allow progress to be made toward human therapy. Analyses of retinal structure and visual function in CEP290-LCA patients were also performed for comparison with the results in the model.
Methods
Rd16;Nrl−/− mice were studied in the first 90 days of life with optical coherence tomography (OCT), electroretinography (ERG), retinal histopathology and immunocytochemistry. Structure and function data from a cohort of patients with CEP290-LCA (n = 15; ages 7–48) were compared with those of the model.
Results
CEP290-LCA patients retain a central island of photoreceptors with normal thickness at the fovea (despite severe visual loss); the extent of this island declined slowly with age. The rd16;Nrl−/− model also showed a relatively slow photoreceptor layer decline in thickness with ∼80% remaining at 3 months. The number of pseudorosettes also became reduced. By comparison to single mutant Nrl−/− mice, UV- and M-cone ERGs of rd16;Nrl−/− were at least 1 log unit reduced at 1 month of age and declined further over the 3 months of monitoring. Expression of GNAT2 and S-opsin also decreased with age.
Conclusions
The natural history of early loss of photoreceptor function with retained cone cell nuclei is common to both CEP290-LCA patients and the rd16;Nrl−/− murine model. Pre-clinical proof-of-concept studies for uniocular therapies would seem most appropriate to begin with intervention at P35–40 and re-study after one month by assaying interocular difference in the UV-cone ERG.
doi:10.1371/journal.pone.0092928
PMCID: PMC3966841  PMID: 24671090
12.  Variations in NPHP5 in Patients With Nonsyndromic Leber Congenital Amaurosis and Senior-Loken Syndrome 
Archives of ophthalmology  2011;129(1):81-87.
Objective
To investigate whether mutations in NPHP5 can cause Leber congenital amaurosis (LCA) without early-onset renal disease.
Methods
DNA samples from 276 individuals with non-syndromic LCA were screened for variations in the NPHP5 gene. Each had been previously screened for mutations in 8 known LCA genes without identifying a disease-causing genotype.
Results
Nine of the 276 LCA probands (3.2%) harbored 2 plausible disease-causing mutations (7 different alleles) in NPHP5. Four of these have been previously reported in patients with Senior-Loken syndrome (F141del, R461X, H506del, and R489X) and 3 are novel (A111del, E346X, and R455X). All 9 patients had severe visual loss from early childhood but none had overt renal disease in the first decade of life. Two patients were diagnosed with nephronophthisis in the second decade. Retinal imaging studies showed retained photoreceptor nuclei and retinal pigment epithelium integrity mainly in the cone-rich central retina, a phenotype with strong similarities to that of NPHP6 disease.
Conclusions
Mutations in NPHP5 can cause LCA without early-onset renal disease. Abnormalities observed in the photoreceptor outer segments (a cilial structure) may explain the severe visual loss in NPHP5-associated LCA.
Clinical Relevance
The persistence of central photoreceptor nuclei despite severe visual loss in NPHP5 disease is encouraging for future therapeutic interventions.
doi:10.1001/archophthalmol.2010.330
PMCID: PMC3952880  PMID: 21220633
13.  Canine Retina Has a Primate Fovea-Like Bouquet of Cone Photoreceptors Which Is Affected by Inherited Macular Degenerations 
PLoS ONE  2014;9(3):e90390.
Retinal areas of specialization confer vertebrates with the ability to scrutinize corresponding regions of their visual field with greater resolution. A highly specialized area found in haplorhine primates (including humans) is the fovea centralis which is defined by a high density of cone photoreceptors connected individually to interneurons, and retinal ganglion cells (RGCs) that are offset to form a pit lacking retinal capillaries and inner retinal neurons at its center. In dogs, a local increase in RGC density is found in a topographically comparable retinal area defined as the area centralis. While the canine retina is devoid of a foveal pit, no detailed examination of the photoreceptors within the area centralis has been reported. Using both in vivo and ex vivo imaging, we identified a retinal region with a primate fovea-like cone photoreceptor density but without the excavation of the inner retina. Similar anatomical structure observed in rare human subjects has been named fovea-plana. In addition, dogs with mutations in two different genes, that cause macular degeneration in humans, developed earliest disease at the newly-identified canine fovea-like area. Our results challenge the dogma that within the phylogenetic tree of mammals, haplorhine primates with a fovea are the sole lineage in which the retina has a central bouquet of cones. Furthermore, a predilection for naturally-occurring retinal degenerations to alter this cone-enriched area fills the void for a clinically-relevant animal model of human macular degenerations.
doi:10.1371/journal.pone.0090390
PMCID: PMC3944008  PMID: 24599007
14.  Comprehensive molecular diagnosis of 179 Leber congenital amaurosis and juvenile retinitis pigmentosa patients by targeted next generation sequencing 
Journal of medical genetics  2013;50(10):674-688.
Background
Leber congenital amaurosis (LCA) and juvenile retinitis pigmentosa (RP) are inherited retinal diseases that cause early onset severe visual impairment. An accurate molecular diagnosis can refine the clinical diagnosis and allow gene specific treatments.
Methods
We developed a capture panel that enriches the exonic DNA of 163 known retinal disease genes. Using this panel, we performed targeted next generation sequencing (NGS) for a large cohort of 179 unrelated and prescreened patients with the clinical diagnosis of LCA or juvenile RP. Systematic NGS data analysis, Sanger sequencing validation, and segregation analysis were utilised to identify the pathogenic mutations. Patients were revisited to examine the potential phenotypic ambiguity at the time of initial diagnosis.
Results
Pathogenic mutations for 72 patients (40%) were identified, including 45 novel mutations. Of these 72 patients, 58 carried mutations in known LCA or juvenile RP genes and exhibited corresponding phenotypes, while 14 carried mutations in retinal disease genes that were not consistent with their initial clinical diagnosis. We revisited patients in the latter case and found that homozygous mutations in PRPH2 can cause LCA/juvenile RP. Guided by the molecular diagnosis, we reclassified the clinical diagnosis in two patients.
Conclusions
We have identified a novel gene and a large number of novel mutations that are associated with LCA/juvenile RP. Our results highlight the importance of molecular diagnosis as an integral part of clinical diagnosis.
doi:10.1136/jmedgenet-2013-101558
PMCID: PMC3932025  PMID: 23847139
15.  AAV-Mediated Gene Therapy in the Guanylate Cyclase (RetGC1/RetGC2) Double Knockout Mouse Model of Leber Congenital Amaurosis 
Human Gene Therapy  2012;24(2):189-202.
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.
Boye and colleagues report that subretinal injection of AAV8 vector carrying the murine retinal guanylate cyclase-1 gene results in restoration of rod and cone function in a mouse model of Leber congenital amaurosis (LCA). Correction was seen at all treatment ages, with functional gains and structural preservation stable for at least 1 year.
doi:10.1089/hum.2012.193
PMCID: PMC3581260  PMID: 23210611
16.  Determining consequences of retinal membrane guanylyl cyclase (RetGC1) deficiency in human Leber congenital amaurosis en route to therapy: residual cone-photoreceptor vision correlates with biochemical properties of the mutants 
Human Molecular Genetics  2012;22(1):168-183.
The GUCY2D gene encodes retinal membrane guanylyl cyclase (RetGC1), a key component of the phototransduction machinery in photoreceptors. Mutations in GUCY2D cause Leber congenital amaurosis type 1 (LCA1), an autosomal recessive human retinal blinding disease. The effects of RetGC1 deficiency on human rod and cone photoreceptor structure and function are currently unknown. To move LCA1 closer to clinical trials, we characterized a cohort of patients (ages 6 months—37 years) with GUCY2D mutations. In vivo analyses of retinal architecture indicated intact rod photoreceptors in all patients but abnormalities in foveal cones. By functional phenotype, there were patients with and those without detectable cone vision. Rod vision could be retained and did not correlate with the extent of cone vision or age. In patients without cone vision, rod vision functioned unsaturated under bright ambient illumination. In vitro analyses of the mutant alleles showed that in addition to the major truncation of the essential catalytic domain in RetGC1, some missense mutations in LCA1 patients result in a severe loss of function by inactivating its catalytic activity and/or ability to interact with the activator proteins, GCAPs. The differences in rod sensitivities among patients were not explained by the biochemical properties of the mutants. However, the RetGC1 mutant alleles with remaining biochemical activity in vitro were associated with retained cone vision in vivo. We postulate a relationship between the level of RetGC1 activity and the degree of cone vision abnormality, and argue for cone function being the efficacy outcome in clinical trials of gene augmentation therapy in LCA1.
doi:10.1093/hmg/dds421
PMCID: PMC3606011  PMID: 23035049
18.  Treatment of Leber Congenital Amaurosis Due to RPE65 Mutations by Ocular Subretinal Injection of Adeno-Associated Virus Gene Vector: Short-Term Results of a Phase I Trial 
Human Gene Therapy  2008;19(10):979-990.
Abstract
Leber congenital amaurosis (LCA) is a group of autosomal recessive blinding retinal diseases that are incurable. One molecular form is caused by mutations in the RPE65 (retinal pigment epithelium-specific 65-kDa) gene. A recombinant adeno-associated virus serotype 2 (rAAV2) vector, altered to carry the human RPE65 gene (rAAV2-CBSB-hRPE65), restored vision in animal models with RPE65 deficiency. A clinical trial was designed to assess the safety of rAAV2-CBSB-hRPE65 in subjects with RPE65-LCA. Three young adults (ages 21–24 years) with RPE65-LCA received a uniocular subretinal injection of 5.96 × 1010 vector genomes in 150 μl and were studied with follow-up examinations for 90 days. Ocular safety, the primary outcome, was assessed by clinical eye examination. Visual function was measured by visual acuity and dark-adapted full-field sensitivity testing (FST); central retinal structure was monitored by optical coherence tomography (OCT). Neither vector-related serious adverse events nor systemic toxicities were detected. Visual acuity was not significantly different from baseline; one patient showed retinal thinning at the fovea by OCT. All patients self-reported increased visual sensitivity in the study eye compared with their control eye, especially noticeable under reduced ambient light conditions. The dark-adapted FST results were compared between baseline and 30–90 days after treatment. For study eyes, sensitivity increases from mean baseline were highly significant (p < 0.001); whereas, for control eyes, sensitivity changes were not significant (p = 0.99). Comparisons are drawn between the present work and two other studies of ocular gene therapy for RPE65-LCA that were carried out contemporaneously and reported.
doi:10.1089/hum.2008.107
PMCID: PMC2940541  PMID: 18774912
19.  Defining the Residual Vision in Leber Congenital Amaurosis Caused by RPE65 Mutations 
PURPOSE
To quantify the residual vision in Leber congenital amaurosis (LCA) caused by RPE65 mutations.
METHODS
Patients with RPE65-LCA (n = 30; ages, 4–55) were studied using electroretinography (ERG), full-field stimulus testing (FST), kinetic and static threshold perimetry, and optical coherence tomography (OCT).
RESULTS
All patients with RPE65-LCA had abnormal ERGs even at the youngest ages. There were no detectable rod ERGs and only reduced cone ERGs. By chromatic FST, however, 59% of patients had measurable rod- and cone-mediated function. The remaining 41% had only cone-mediated function. Extent of kinetic fields varied widely in the first two decades of life but, by the end of the third decade, there was very little measurable field. Regional patterns of visual loss were evident using dark-adapted static threshold perimetry. The mildest dysfunctions showed relatively homogeneous sensitivity loss beyond the central field. Mid-peripheral dysfunction was a later feature; finally, only central and peripheral islands remained. Colocalized measures of visual function and retinal structure by OCT showed that visual function was detectable when a photoreceptor layer was detectable.
CONCLUSIONS
Residual rod as well as cone function is detectable in RPE65-LCA. The finding of different regional patterns of visual loss in these patients suggests that the optimal retinal site(s) for subretinal gene delivery to achieve efficacy are likely to change with disease progression.
doi:10.1167/iovs.08-2696
PMCID: PMC2731629  PMID: 19117922
20.  Photoreceptor Layer Topography in Children with Leber Congenital Amaurosis Caused by RPE65 Mutations 
PURPOSE
To study the topography of photoreceptor loss early in the course of Leber congenital amaurosis (LCA) caused by RPE65 mutations.
METHODS
Young patients with RPE65-LCA (n = 9; ages, 6–17 years) were studied with optical coherence tomography (OCT) in a wide region of central retina. Outer nuclear layer (ONL) thickness was mapped topographically and compared with that in normal subjects and in older patients with RPE65-LCA.
RESULTS
Photoreceptor layer topography was abnormal in all young patients with RPE65-LCA. Foveal and extrafoveal ONL was reduced in most patients. There were interindividual differences, with ONL thicknesses at most retinal locations ranging from near the detectability limit to a significant fraction of normal. These differences were not clearly related to age. In most patients, there was a thinner ONL inferior to the fovea compared with that in the superior retina. Summary maps obtained by aligning and averaging photoreceptor topography across all young patients showed a relative preservation of ONL in the superior-temporal and temporal pericentral retina. These retinal regions also showed the greatest magnitude of interindividual variation.
CONCLUSIONS
Photoreceptor loss in the foveal and extrafoveal retina was prominent, even in the youngest patients studied. Differences in the topography of residual photoreceptors in children with RPE65-LCA suggest that it may be advisable to use individualized ONL mapping to guide the location of sub-retinal injections for gene therapy and thereby maximize the potential for efficacy.
doi:10.1167/iovs.08-2121
PMCID: PMC2731624  PMID: 18539930
21.  Leber congenital amaurosis caused by Lebercilin (LCA5) mutation: Retained photoreceptors adjacent to retinal disorganization 
Molecular Vision  2009;15:1098-1106.
Purpose
To determine the retinal disease expression in the rare form of Leber congenital amaurosis (LCA) caused by Lebercilin (LCA5) mutation.
Methods
Two young unrelated LCA patients, ages six years (P1) and 25 years (P2) at last visit, both with the same homozygous mutation in the LCA5 gene, were evaluated clinically and with noninvasive studies. En face imaging was performed with near-infrared (NIR) reflectance and autofluorescence (AF); cross-sectional retinal images were obtained with optical coherence tomography (OCT). Dark-adapted thresholds were measured in the older patient; and the transient pupillary light reflex was recorded and quantified in both patients.
Results
Both LCA5 patients had light perception vision only, hyperopia, and nystagmus. P1 showed a prominent central island of retinal pigment epithelium (RPE) surrounded by alternating elliptical-appearing areas of decreased and increased pigmentation. Retinal laminar architecture at and near the fovea was abnormal in both patients. Foveal outer nuclear layer (ONL) was present in P1 and P2 but to different degrees. With increasing eccentricity, there was retinal laminar disorganization. Regions of pericentral and midperipheral retina in P1, but not P2, could retain measurable ONL and less laminopathy. P2 had a small central island of perception with >5 log units of sensitivity loss. Pupillary responsiveness was present in both LCA5 patients; the thresholds were abnormally elevated by ≥5.5 log units.
Conclusions
LCA5 patients had evidence of retained photoreceptors mainly in the central retina. Retinal remodeling was present in pericentral regions in both patients. The NIR reflectance and NIR-AF imaging in the younger patient suggested preserved RPE in retinal regions with retained photoreceptors. Detailed phenotype studies in other LCA5 patients with longitudinal follow-up will help determine the feasibility of future intervention in this rare disease.
PMCID: PMC2690955  PMID: 19503738
22.  Non-exomic and synonymous variants in ABCA4 are an important cause of Stargardt disease 
Human Molecular Genetics  2013;22(25):5136-5145.
Mutations in ABCA4 cause Stargardt disease and other blinding autosomal recessive retinal disorders. However, sequencing of the complete coding sequence in patients with clinical features of Stargardt disease sometimes fails to detect one or both mutations. For example, among 208 individuals with clear clinical evidence of ABCA4 disease ascertained at a single institution, 28 had only one disease-causing allele identified in the exons and splice junctions of the primary retinal transcript of the gene. Haplotype analysis of these 28 probands revealed 3 haplotypes shared among ten families, suggesting that 18 of the 28 missing alleles were rare enough to be present only once in the cohort. We hypothesized that mutations near rare alternate splice junctions in ABCA4 might cause disease by increasing the probability of mis-splicing at these sites. Next-generation sequencing of RNA extracted from human donor eyes revealed more than a dozen alternate exons that are occasionally incorporated into the ABCA4 transcript in normal human retina. We sequenced the genomic DNA containing 15 of these minor exons in the 28 one-allele subjects and observed five instances of two different variations in the splice signals of exon 36.1 that were not present in normal individuals (P < 10−6). Analysis of RNA obtained from the keratinocytes of patients with these mutations revealed the predicted alternate transcript. This study illustrates the utility of RNA sequence analysis of human donor tissue and patient-derived cell lines to identify mutations that would be undetectable by exome sequencing.
doi:10.1093/hmg/ddt367
PMCID: PMC3842174  PMID: 23918662
23.  Visual Acuity Changes in Patients with Leber Congenital Amaurosis (LCA) and Mutations in the CEP290 Gene 
JAMA ophthalmology  2013;131(2):178-182.
Objective
To evaluate changes in visual acuity (VA) over time in patients with Leber congenital amaurosis (LCA) and mutations in the CEP290 gene.
Patients
Forty-three patients with LCA and CEP290 mutations participated.
Methods
VA was determined at the initial and most recent visits.
Main Outcome Measures
The best-corrected VA at the initial and most recent visits, and the correlation between age and VA.
Results
At the initial visit, 14 patients had measurable chart VA in the better-seeing eye, 25 patients had non-measurable chart VA, and VA was not assessed in 4 young patients. At the most recent visit, 15 patients had measurable chart VA and 28 had non-measurable chart VA. The average duration between the two visits was 10.4 years (range: 2 to 47 years). For patients with measurable chart VA, the median logMAR value at the initial visit, 0.75 (range: 0.10 to 2.30), and most recent visit, 0.70 (range: 0.10 to 2.00), did not differ significantly (p > 0.05). There was no significant relationship between VA and age.
Conclusions
Patients with LCA and CEP290 mutations had a wide spectrum of VA that was not related to age or length of follow-up. Severe VA loss was observed in most, but not all, patients in the first decade. These data will help clinicians provide counseling on VA changes in patients with CEP290 mutations and could be of value for future treatment trials.
doi:10.1001/2013.jamaophthalmol.354
PMCID: PMC3688627  PMID: 23411883
24.  Mutations in RPGR and RP2 Account for 15% of Males with Simplex Retinal Degenerative Disease 
Purpose.
To determine the proportion of male patients presenting simplex retinal degenerative disease (RD: retinitis pigmentosa [RP] or cone/cone-rod dystrophy [COD/CORD]) with mutations in the X-linked retinal degeneration genes RPGR and RP2.
Methods.
Simplex males were defined as patients with no known affected family members. Patients were excluded if they had a family history of parental consanguinity. Blood samples from a total of 214 simplex males with a diagnosis of retinal degeneration were collected for genetic analysis. The patients were screened for mutations in RPGR and RP2 by direct sequencing of PCR-amplified genomic DNA.
Results.
We identified pathogenic mutations in 32 of the 214 patients screened (15%). Of the 29 patients with a diagnosis of COD/CORD, four mutations were identified in the ORF15 mutational hotspot of the RPGR gene. Of the 185 RP patients, three patients had mutations in RP2 and 25 had RPGR mutations (including 12 in the ORF15 region).
Conclusions.
This study represents mutation screening of RPGR and RP2 in the largest cohort, to date, of simplex males affected with RP or COD/CORD. Our results demonstrate a substantial contribution of RPGR mutations to retinal degenerations, and in particular, to simplex RP. Based on our findings, we suggest that RPGR should be considered as a first tier gene for screening isolated males with retinal degeneration.
Identification of mutations in 15% of the screened patients has important implications for guiding clinicians who are ordering genetic testing and provides a strong argument for screening the RPGR gene in simplex cases of retinal degenerative diseases.
doi:10.1167/iovs.12-11025
PMCID: PMC3522443  PMID: 23150612
25.  Macular Function in Macular Degenerations: Repeatability of Microperimetry as a Potential Outcome Measure for ABCA4-Associated Retinopathy Trials 
Microperimetry is often used to measure macular function in patients with unstable fixation, but repeatability is not well established. This study shows that microperimetric sensitivities can be obtained with a predictable reliability at individual retinal loci independent of disease severity.
Purpose.
To measure macular visual function in patients with unstable fixation, to define the photoreceptor source of this function, and to estimate its test-retest repeatability as a prerequisite to clinical trials.
Methods.
Patients (n = 38) with ABCA4-associated retinal degeneration (RD) or with retinitis pigmentosa (RP) were studied with retina-tracking microperimetry along the foveo-papillary profile between the fovea and the optic nerve head, and point-by-point test-retest repeatability was estimated. A subset with foveal fixation was also studied with dark-adapted projection perimetry using monochromatic blue and red stimuli along the horizontal meridian.
Results.
Macular function in ABCA4-RD patients transitioned from lower sensitivity at the parafovea to higher sensitivity in the perifovea. RP patients had the inverse pattern. Red-on-red microperimetric sensitivities successfully avoided ceiling effects and were highly correlated with absolute sensitivities. Point-by-point test-retest limits (95% confidence intervals) were ±4.2 dB; repeatability was not related to mean sensitivity, eccentricity from the fovea, age, fixation location, or instability. Repeatability was also not related to the local slope of sensitivity and was unchanged in the parapapillary retina.
Conclusions.
Microperimetry allows reliable testing of macular function in RD patients without foveal fixation in longitudinal studies evaluating natural disease progression or efficacy of therapeutic trials. A single estimate of test-retest repeatability can be used to determine significant changes in visual function at individual retinal loci within diseased regions that are homogeneous and those that are heterogeneous and also in transition zones at high risk for disease progression.
doi:10.1167/iovs.11-8415
PMCID: PMC3317423  PMID: 22247458

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