Safety and Vision Outcomes of Subretinal Gene Therapy Targeting Cone Photoreceptors in Achromatopsia: A Nonrandomized Controlled Trial.

Importance: Achromatopsia linked to variations in the CNGA3 gene is associated with day blindness, poor visual acuity, photophobia, and involuntary eye movements owing to lack of cone photoreceptor function. No treatment is currently available. Objective: To assess safety and vision outcomes of supplemental gene therapy with adeno-associated virus (AAV) encoding CNGA3 (AAV8.CNGA3) in patients with CNGA3-linked achromatopsia. Design, Setting, and Participants: This open-label, exploratory nonrandomized controlled trial tested safety and vision outcomes of gene therapy vector AAV8.CNGA3 administered by subretinal injection at a single center. Nine patients (3 per dose group) with a clinical diagnosis of achromatopsia and confirmed biallelic disease-linked variants in CNGA3 were enrolled between November 5, 2015, and September 22, 2016. Data analysis was performed from June 6, 2017, to March 12, 2018. Intervention: Patients received a single unilateral injection of 1.0 × 1010, 5.0 × 1010, or 1.0 × 1011 total vector genomes of AAV8.CNGA3 and were followed up for a period of 12 months (November 11, 2015, to October 10, 2017). Main Outcomes and Measures: Safety as the primary end point was assessed by clinical examination of ocular inflammation. Systemic safety was assessed by vital signs, routine clinical chemistry testing, and full and differential blood cell counts. Secondary outcomes were change in visual function from baseline in terms of spatial and temporal resolution and chromatic, luminance, and contrast sensitivity throughout a period of 12 months after treatment. Results: Nine patients (mean [SD] age, 39.6 [11.9] years; age range, 24-59 years; 8 [89%] male) were included in the study. Baseline visual acuity letter score (approximate Snellen equivalent) ranged from 34 (20/200) to 49 (20/100), whereas baseline contrast sensitivity log scores ranged from 0.1 to 0.9. All 9 patients underwent surgery and subretinal injection of AAV8.CNGA3 without complications. No substantial safety problems were observed during the 12-month follow-up period. Despite the congenital deprivation of cone photoreceptor-mediated vision in achromatopsia, all 9 treated eyes demonstrated some level of improvement in secondary end points regarding cone function, including mean change in visual acuity of 2.9 letters (95% CI, 1.65-4.13; P = .006, 2-sided t test paired samples). Contrast sensitivity improved by a mean of 0.33 log (95% CI, 0.14-0.51 log; P = .003, 2-sided t test paired samples). Conclusions and Relevance: Subretinal gene therapy with AAV8.CNGA3 was not associated with substantial safety problems and was associated with cone photoreceptor activation in adult patients, as reflected by visual acuity and contrast sensitivity gains. Trial Registration: ClinicalTrials.gov Identifier: NCT02610582.

A retinal model of cerebral malaria.

Malaria is a causative factor in about 500.000 deaths each year world-wide. Cerebral malaria is a particularly severe complication of this disease and thus associated with an exceedingly high mortality. Malaria retinopathy is an ocular manifestation often associated with cerebral malaria, and presumably shares a substantial part of its pathophysiology. Here, we describe that indeed murine malaria retinopathy reproduced the main hallmarks of the corresponding human disease. In the living animal, we were able to follow the circulation and cellular localization of malaria parasites transgenically labelled with GFP via non-invasive in vivo retinal imaging. We found that malaria parasites cross the blood-retinal-barrier and infiltrate the neuroretina, concomitant with an extensive, irreversible, and long-lasting retinal neurodegeneration. Furthermore, anti-malarial treatment with dihydroartemisinin strongly diminished the load of circulating parasites but resolved the symptoms of the retinopathy only in part. In summary, we introduce here a novel preclinical model for human cerebral malaria that is much more directly accessible for studies into disease pathophysiology and development of novel treatment approaches. In vivo retinal imaging may furthermore serve as a valuable tool for the early diagnosis of the human disease.

Cystoid edema, neovascularization and inflammatory processes in the murine Norrin-deficient retina.

Mutations in the Norrin (NDP) gene cause severe developmental blood vessel defects in the retina leading to congenital blindness. In the retina of Ndph-knockout mice only the superficial capillary network develops. Here, a detailed characterization of this mouse model at late stages of the disease using in vivo retinal imaging revealed cystoid structures that closely resemble the ovoid cysts in the inner nuclear layer of the human retina with cystoid macular edema (CME). In human CME an involvement of Müller glia cells is hypothesized. In Ndph-knockout retinae we could demonstrate that activated Müller cells were located around and within these cystoid spaces. In addition, we observed extensive activation of retinal microglia and development of neovascularization. Furthermore, ex vivo analyses detected extravasation of monocytic cells suggesting a breakdown of the blood retina barrier. Thus, we could demonstrate that also in the developmental retinal vascular pathology present in the Ndph-knockout mouse inflammatory processes are active and may contribute to further retinal degeneration. This observation delivers a new perspective for curative treatments of retinal vasculopathies. Modulation of inflammatory responses might reduce the symptoms and improve visual acuity in these diseases.

Gene Therapy Successfully Delays Degeneration in a Mouse Model of PDE6A-Linked Retinitis Pigmentosa (RP43).

Retinitis pigmentosa type 43 (RP43) is a blinding disease caused by mutations in the gene for rod phosphodiesterase 6 alpha (PDE6A). The disease process begins with a dysfunction of rod photoreceptors, subsequently followed by a currently untreatable progressive degeneration of the entire outer retina. Aiming at a curative approach via PDE6A gene supplementation, a novel adeno-associated viral (AAV) vector was developed for expression of the human PDE6A cDNA under control of the human rhodopsin promotor (rAAV8.PDE6A). This study assessed the therapeutic efficacy of rAAV8.PDE6A in the Pde6anmf363/nmf363-mutant mouse model of RP43. All mice included in this study were treated with sub-retinal injections of the vector at 2 weeks after birth. The therapeutic effect was monitored at 1 month and 6 months post injection. Biological function of the transgene was assessed in vivo by means of electroretinography. The degree of morphological rescue was investigated both in vivo using optical coherence tomography and ex vivo by immunohistological staining. It was found that the novel rAAV8.PDE6A vector resulted in a stable and efficient expression of PDE6A protein in rod photoreceptors of Pde6anmf363/nmf363 mice following treatment at both the short- and long-term time points. The treatment led to a substantial morphological preservation of outer nuclear layer thickness, rod outer segment structure, and prolonged survival of cone photoreceptors for at least 6 months. Additionally, the ERG analysis confirmed a restoration of retinal function in a group of treated mice. Taken together, this study provides successful proof-of-concept for the cross-species efficacy of the rAAV8.PDE6A vector developed for use in human patients. Importantly, the data show stable expression and rescue effects for a prolonged period of time, raising hope for future translational studies based on this approach.

AAV-Mediated Gene Supplementation Therapy in Achromatopsia Type 2: Preclinical Data on Therapeutic Time Window and Long-Term Effects.

Achromatopsia type 2 (ACHM2) is a severe, inherited eye disease caused by mutations in the CNGA3 gene encoding the α subunit of the cone photoreceptor cyclic nucleotide-gated (CNG) channel. Patients suffer from strongly impaired daylight vision, photophobia, nystagmus, and lack of color discrimination. We have previously shown in the Cnga3 knockout (KO) mouse model of ACHM2 that gene supplementation therapy is effective in rescuing cone function and morphology and delaying cone degeneration. In our preclinical approach, we use recombinant adeno-associated virus (AAV) vector-mediated gene transfer to express the murine Cnga3 gene under control of the mouse blue opsin promoter. Here, we provide novel data on the efficiency and permanence of such gene supplementation therapy in Cnga3 KO mice. Specifically, we compare the influence of two different AAV vector capsids, AAV2/5 (Y719F) and AAV2/8 (Y733F), on restoration of cone function, and assess the effect of age at time of treatment on the long-term outcome. The evaluation included in vivo analysis of retinal function using electroretinography (ERG) and immunohistochemical analysis of vector-driven Cnga3 transgene expression. We found that both vector capsid serotypes led to a comparable rescue of cone function over the observation period between 4 weeks and 3 months post treatment. In addition, a clear therapeutic effect was present in mice treated at 2 weeks of age as well as in mice treated at 3 months of age at the first assessment at 4 weeks after treatment. Importantly, the effect extended in both cases over the entire observation period of 12 months post treatment. However, the average ERG amplitude levels differed between the two groups, suggesting a role of the absolute age, or possibly, the associated state of the degeneration, on the achievable outcome. In summary, we found that the therapeutic time window of opportunity for AAV-mediated Cnga3 gene supplementation therapy in the Cnga3 KO mouse model extends at least to an age of 3 months, but is presumably limited by the condition, number and topographical distribution of remaining cones at the time of treatment. No impact of the choice of capsid on the therapeutic success was detected.

Long-term consequences of developmental vascular defects on retinal vessel homeostasis and function in a mouse model of Norrie disease.

Loss of Norrin signalling due to mutations in the Norrie disease pseudoglioma gene causes severe vascular defects in the retina, leading to visual impairment and ultimately blindness. While the emphasis of experimental work so far was on the developmental period, we focus here on disease mechanisms that induce progression into severe adult disease. The goal of this study was the comprehensive analysis of the long-term effects of the absence of Norrin on vascular homeostasis and retinal function. In a mouse model of Norrie disease retinal vascular morphology and integrity were studied by means of in vivo angiography; the vascular constituents were assessed in detailed histological analyses using quantitative retinal morphometry. Finally, electroretinographic analyses were performed to assess the retinal function in adult Norrin deficient animals. We could show that the primary developmental defects not only persisted but developed into further vascular abnormalities and microangiopathies. In particular, the overall vessel homeostasis, the vascular integrity, and also the cellular constituents of the vascular wall were affected in the adult Norrin deficient retina. Moreover, functional analyses indicated to persistent hypoxia in the neural retina which was suggested as one of the major driving forces of disease progression. In summary, our data provide evidence that the key to adult Norrie disease are ongoing vascular modifications, driven by the persistent hypoxic conditions, which are ineffective to compensate for the primary Norrin-dependent defects.

Loss of HCN1 enhances disease progression in mouse models of CNG channel-linked retinitis pigmentosa and achromatopsia.

Most inherited blinding diseases are characterized by compromised retinal function and progressive degeneration of photoreceptors. However, the factors that affect the life span of photoreceptors in such degenerative retinal diseases are rather poorly understood. Here, we explore the role of hyperpolarization-activated cyclic nucleotide-gated channel 1 (HCN1) in this context. HCN1 is known to adjust retinal function under mesopic conditions, and although it is expressed at high levels in rod and cone photoreceptor inner segments, no association with any retinal disorder has yet been found. We investigated the effects of an additional genetic deletion of HCN1 on the function and survival of photoreceptors in a mouse model of CNGB1-linked retinitis pigmentosa (RP). We found that the absence of HCN1 in Cngb1 knockout (KO) mice exacerbated photoreceptor degeneration. The deleterious effect was reduced by expression of HCN1 using a viral vector. Moreover, pharmacological inhibition of HCN1 also enhanced rod degeneration in Cngb1 KO mice. Patch-clamp recordings revealed that the membrane potentials of Cngb1 KO and Cngb1/Hcn1 double-KO rods were both significantly depolarized. We also found evidence for altered calcium homeostasis and increased activation of the protease calpain in Cngb1/Hcn1 double-KO mice. Finally, the deletion of HCN1 also exacerbated degeneration of cone photoreceptors in a mouse model of CNGA3-linked achromatopsia. Our results identify HCN1 as a major modifier of photoreceptor degeneration and suggest that pharmacological inhibition of HCN channels may enhance disease progression in RP and achromatopsia patients.

HCN1 Channels Enhance Rod System Responsivity in the Retina under Conditions of Light Exposure.

PURPOSE: Vision originates in rods and cones at the outer retina. Already at these early stages, diverse processing schemes shape and enhance image information to permit perception over a wide range of lighting conditions. In this work, we address the role of hyperpolarization-activated and cyclic nucleotide-gated channels 1 (HCN1) in rod photoreceptors for the enhancement of rod system responsivity under conditions of light exposure. METHODS: To isolate HCN1 channel actions in rod system responses, we generated double mutant mice by crossbreeding Hcn1-/- mice with Cnga3-/- mice in which cones are non-functional. Retinal function in the resulting Hcn1-/- Cnga3-/- animals was followed by means of electroretinography (ERG) up to the age of four month. Retinal imaging via scanning laser ophthalmoscopy (SLO) and optical coherence tomography (OCT) was also performed to exclude potential morphological alterations. RESULTS: This study on Hcn1-/- Cnga3-/- mutant mice complements our previous work on HCN1 channel function in the retina. We show here in a functional rod-only setting that rod responses following bright light exposure terminate without the counteraction of HCN channels much later than normal. The resulting sustained signal elevation does saturate the retinal network due to an intensity-dependent reduction in the dynamic range. In addition, the lack of rapid adaptational feedback modulation of rod photoreceptor output via HCN1 in this double mutant limits the ability to follow repetitive (flicker) stimuli, particularly under mesopic conditions. CONCLUSIONS: This work corroborates the hypothesis that, in the absence of HCN1-mediated feedback, the amplitude of rod signals remains at high levels for a prolonged period of time, leading to saturation of the retinal pathways. Our results demonstrate the importance of HCN1 channels for regular vision.

Retinitis pigmentosa: impact of different Pde6a point mutations on the disease phenotype.

Mutations in the PDE6A gene can cause rod photoreceptors degeneration and the blinding disease retinitis pigmentosa (RP). While a number of pathogenic PDE6A mutations have been described, little is known about their impact on compound heterozygous situations and potential interactions of different disease-causing alleles. Here, we used a novel mouse model for the Pde6a R562W mutation in combination with an existing line carrying the V685M mutation to generate compound heterozygous Pde6a V685M/R562W animals, exactly homologous to a case of human RP. We compared the progression of photoreceptor degeneration in these compound heterozygous mice with the homozygous V685M and R562W mutants, and additionally with the D670G line that is known for a relatively mild phenotype. We investigated PDE6A expression, cyclic guanosine mono-phosphate accumulation, calpain and caspase activity, in vivo retinal function and morphology, as well as photoreceptor cell death and survival. This analysis confirms the severity of different Pde6a mutations and indicates that compound heterozygous mutants behave like intermediates of the respective homozygous situations. Specifically, the severity of the four different Pde6a situations may be categorized by the pace of photoreceptor degeneration: V685M (fastest) > V685M/R562W > R562W > D670G (slowest). While calpain activity was strongly increased in all four mutants, caspase activity was not. This points to the execution of non-apoptotic cell death and may lead to the identification of new targets for therapeutic interventions. For individual RP patients, our study may help to predict time-courses for Pde6a-related retinal degeneration and thereby facilitate the definition of a window-of-opportunity for clinical interventions.

Mutations in the unfolded protein response regulator ATF6 cause the cone dysfunction disorder achromatopsia.

Achromatopsia (ACHM) is an autosomal recessive disorder characterized by color blindness, photophobia, nystagmus and severely reduced visual acuity. Using homozygosity mapping and whole-exome and candidate gene sequencing, we identified ten families carrying six homozygous and two compound-heterozygous mutations in the ATF6 gene (encoding activating transcription factor 6A), a key regulator of the unfolded protein response (UPR) and cellular endoplasmic reticulum (ER) homeostasis. Patients had evidence of foveal hypoplasia and disruption of the cone photoreceptor layer. The ACHM-associated ATF6 mutations attenuate ATF6 transcriptional activity in response to ER stress. Atf6(-/-) mice have normal retinal morphology and function at a young age but develop rod and cone dysfunction with increasing age. This new ACHM-related gene suggests a crucial and unexpected role for ATF6A in human foveal development and cone function and adds to the list of genes that, despite ubiquitous expression, when mutated can result in an isolated retinal photoreceptor phenotype.

Scale Adjustments to Facilitate Two-Dimensional Measurements in OCT Images.

PURPOSE: To address the problem of unequal scales for the measurement of two-dimensional structures in OCT images, and demonstrate the use of intra¬ocular objects of known dimensions in the murine eye for the equal calibration of axes. METHODS: The first part of this work describes the mathematical foundation of major distortion effects introduced by X-Y scaling differences. Illustrations were generated with CorelGraph X3 software. The second part bases on image data obtained with a HRA2 Spectralis (Heidelberg Engineering) in SV129 wild-type mice. Subretinally and intravitreally implanted microbeads, alginate capsules with a diameter of 154±5 µm containing GFP-marked mesenchymal stem cells (CellBeads), were used as intraocular objects for calibration. RESULTS: The problems encountered with two-dimensional measurements in cases of unequal scales are demonstrated and an estimation of the resulting errors is provided. Commonly, the Y axis is reliably calibrated using outside standards like histology or manufacturer data. We show here that intraocular objects like dimensionally stable spherical alginate capsules allow for a two-dimensional calibration of the acquired OCT raw images by establishing a relation between X and Y axis data. For our setup, a correction factor of about 3.3 was determined using both epiretinally and subretinally positioned beads (3.350 ± 0.104 and 3.324 ± 0.083, respectively). CONCLUSIONS: In this work, we highlight the distortion-related problems in OCT image analysis induced by unequal X and Y scales. As an exemplary case, we provide data for a two-dimensional in vivo OCT image calibration in mice using intraocular alginate capsules. Our results demonstrate the need for a proper two-dimensional calibration of OCT data, and we believe that equal scaling will certainly improve the efficiency of OCT image analysis.

VEGF Mediates ApoE4-Induced Neovascularization and Synaptic Pathology in the Choroid and Retina.

Apolipoprotein E4 (apoE4), the most prevalent genetic risk factor for Alzheimer's disease (AD), is associated with neuronal and vascular impairments. Recent findings suggest that retina of apoE4 mice have synaptic and functional impairments. We presently investigated the effects of apoE4 on retinal and choroidal vasculature and the possible role of VEGF in these effects. There were no histological differences between the retinal and choroidal vasculatures of naïve apoE3 and apoE4 mice. In contrast, laserdriven choroidal injury induced higher levels of choroidal neovascularization (CNV) in apoE4 than in apoE3 mice. These effects were associated with an inflammatory response and with activation of the Muller cells and asrocytic markers gluthatione synthetase and GFAP, all of which were more pronounced in the apoE4 mice. CNV also induced a transient increase in the levels of the synaptic markers synaptophysin and PSD95 which were however similar in the apoE4 and apoE3 naive mice. Retinal and choroidal VEGF and apoE levels were lower in naïve apoE4 than in corresponding apoE3 mice. In contrast, VEGF and apoE levels rose more pronouncedly following laser injury in the apoE4 than in apoE3 mice. Taken together, these findings suggest that the apoE4-induced retinal impairments, under basal conditions, may be related to reduced VEGF levels in the eyes of these mice. The hyper-neovascularization in the apoE4 mice might be driven by increased inflammation and the associated surge in VEGF following injury. Retinal and choroidal VEGF and apoE levels were lower in naïve apoE4 than in corresponding apoE3 mice. In contrast, VEGF and apoE levels rose more pronouncedly following laser injury in the apoE4 than in apoE3 mice. Taken together, these findings suggest that the apoE4-induced retinal impairments, under basal conditions, may be related to reduced VEGF levels in the eyes of these mice. The hyper-neovascularization in the apoE4 mice might be driven by increased inflammation and the associated surge in VEGF following injury.

Influence of the ß2-Subunit of L-Type Voltage-Gated Cav Channels on the Structural and Functional Development of Photoreceptor Ribbon Synapses.

PURPOSE: The cacnb2 gene encodes the ß2 subunit (Cavß2) of voltage-gated Ca2+ channels in photoreceptors, and its targeted deletion in mice has previously been shown to cause altered retinal morphology and synaptic transmission. The purpose of this study was to provide a detailed morphologic study combined with experiments on the altered functions of photoreceptor ribbon synapses lacking Cavß2. METHODS: A cacnb2-deficient mouse strain was generated and deletion of the Cavß2 in the retina documented by biochemical and immunhistochemical approaches. Ultrastructural changes of photoreceptor ribbon synapses were examined by electronmicroscopy and functional implications of the lack of Cavß2 studied by depolarization-induced Ca2+ influx into isolated photoreceptor cells and electroretinography. RESULTS: Voltage-gated Ca2+ influx into rod photoreceptors lacking Cavß2 was abolished and the typical rod ribbon-type active zones were absent in Cavß2-deficient retinas. The active zone and the architecture of the presynaptic terminals were severely altered in rod synapses. Cone photoreceptor and the bipolar cell ribbon synapses were largely spared from ultrastructural changes although peanut agglutinin (PNA) labelling and photopic ERG analyses demonstrated that also cone pathways were disturbed in Cavß2-deficient retinas. CONCLUSIONS: The presence of the Cavß2 is essential for the structural integrity and function of the rod photoreceptor synapse. The Cavß2 is less essential for the morphology of cone and bipolar cell ribbon synapses, although the impaired photopic electroretinogram suggests a functional alteration also of the cone-mediated signaling in Cavß2-deficient retinas.

Elk3 deficiency causes transient impairment in post-natal retinal vascular development and formation of tortuous arteries in adult murine retinae.

Serum Response Factor (SRF) fulfills essential roles in post-natal retinal angiogenesis and adult neovascularization. These functions have been attributed to the recruitment by SRF of the cofactors Myocardin-Related Transcription Factors MRTF-A and -B, but not the Ternary Complex Factors (TCFs) Elk1 and Elk4. The role of the third TCF, Elk3, remained unknown. We generated a new Elk3 knockout mouse line and showed that Elk3 had specific, non-redundant functions in the retinal vasculature. In Elk3(-/-) mice, post-natal retinal angiogenesis was transiently delayed until P8, after which it proceeded normally. Interestingly, tortuous arteries developed in Elk3(-/-) mice from the age of four weeks, and persisted into late adulthood. Tortuous vessels have been observed in human pathologies, e.g. in ROP and FEVR. These human disorders were linked to altered activities of vascular endothelial growth factor (VEGF) in the affected eyes. However, in Elk3(-/-) mice, we did not observe any changes in VEGF or several other potential confounding factors, including mural cell coverage and blood pressure. Instead, concurrent with the post-natal transient delay of radial outgrowth and the formation of adult tortuous arteries, Elk3-dependent effects on the expression of Angiopoietin/Tie-signalling components were observed. Moreover, in vitro microvessel sprouting and microtube formation from P10 and adult aortic ring explants were reduced. Collectively, these results indicate that Elk3 has distinct roles in maintaining retinal artery integrity. The Elk3 knockout mouse is presented as a new animal model to study retinal artery tortuousity in mice and human patients.

Towards a quantitative OCT image analysis.

BACKGROUND: Optical coherence tomography (OCT) is an invaluable diagnostic tool for the detection and follow-up of retinal pathology in patients and experimental disease models. However, as morphological structures and layering in health as well as their alterations in disease are complex, segmentation procedures have not yet reached a satisfactory level of performance. Therefore, raw images and qualitative data are commonly used in clinical and scientific reports. Here, we assess the value of OCT reflectivity profiles as a basis for a quantitative characterization of the retinal status in a cross-species comparative study. METHODS: Spectral-Domain Optical Coherence Tomography (OCT), confocal Scanning-Laser Ophthalmoscopy (SLO), and Fluorescein Angiography (FA) were performed in mice (Mus musculus), gerbils (Gerbillus perpadillus), and cynomolgus monkeys (Macaca fascicularis) using the Heidelberg Engineering Spectralis system, and additional SLOs and FAs were obtained with the HRA I (same manufacturer). Reflectivity profiles were extracted from 8-bit greyscale OCT images using the ImageJ software package (http://rsb.info.nih.gov/ij/). RESULTS: Reflectivity profiles obtained from OCT scans of all three animal species correlated well with ex vivo histomorphometric data. Each of the retinal layers showed a typical pattern that varied in relative size and degree of reflectivity across species. In general, plexiform layers showed a higher level of reflectivity than nuclear layers. A comparison of reflectivity profiles from specialized retinal regions (e.g. visual streak in gerbils, fovea in non-human primates) with respective regions of human retina revealed multiple similarities. In a model of Retinitis Pigmentosa (RP), the value of reflectivity profiles for the follow-up of therapeutic interventions was demonstrated. CONCLUSIONS: OCT reflectivity profiles provide a detailed, quantitative description of retinal layers and structures including specialized retinal regions. Our results highlight the potential of this approach in the long-term follow-up of therapeutic strategies.

Gene therapy restores vision and delays degeneration in the CNGB1(-/-) mouse model of retinitis pigmentosa.

Retinitis pigmentosa (RP) is a severe retinal disease characterized by a progressive degeneration of rod photoreceptors and a secondary loss of cone function. Here, we used CNGB1-deficient (CNGB1(-/-)) mice, a mouse model for autosomal recessive RP, to evaluate the efficacy of adeno-associated virus (AAV) vector-mediated gene therapy for the treatment of RP. The treatment restored normal expression of rod CNG channels and rod-driven light responses in the CNGB1(-/-) retina. This led to a substantial delay of retinal degeneration and long-term preservation of retinal morphology. Finally, treated CNGB1(-/-) mice performed significantly better than untreated mice in a rod-dependent vision-guided behavior test. In summary, this study holds promise for the treatment of rod channelopathy-associated retinitis pigmentosa by AAV-mediated gene replacement.

CRB2 acts as a modifying factor of CRB1-related retinal dystrophies in mice.

Mutations in the CRB1 gene lead to retinal dystrophies ranging from Leber congenital amaurosis (LCA) to early-onset retinitis pigmentosa (RP), due to developmental defects or loss of adhesion between photoreceptors and Müller glia cells, respectively. Whereas over 150 mutations have been found, no clear genotype-phenotype correlation has been established. Mouse Crb1 knockout retinas show a mild phenotype limited to the inferior quadrant, whereas Crb2 knockout retinas display a severe degeneration throughout the retina mimicking the phenotype observed in RP patients associated with CRB1 mutations. Crb1Crb2 double mutant retinas have severe developmental defects similar to the phenotype observed in LCA patients associated with CRB1 mutations. Therefore, CRB2 is a candidate modifying gene of human CRB1-related retinal dystrophy. In this study, we studied the cellular localization of CRB1 and CRB2 in human retina and tested the influence of the Crb2 gene allele on Crb1-retinal dystrophies in mice. We found that in contrast to mice, in the human retina CRB1 protein was expressed at the subapical region in photoreceptors and Müller glia cells, and CRB2 only in Müller glia cells. Genetic ablation of one allele of Crb2 in heterozygote Crb1(+/-) retinas induced a mild retinal phenotype, but in homozygote Crb1 knockout mice lead to an early and severe phenotype limited to the entire inferior retina. Our data provide mechanistic insight for CRB1-related LCA and RP.

Targeted ablation of Crb2 in photoreceptor cells induces retinitis pigmentosa.

In humans, the Crumbs homolog-1 (CRB1) gene is mutated in autosomal recessive Leber congenital amaurosis and early-onset retinitis pigmentosa. In mammals, the Crumbs family is composed of: CRB1, CRB2, CRB3A and CRB3B. Recently, we showed that removal of mouse Crb2 from retinal progenitor cells, and consequent removal from Müller glial and photoreceptor cells, results in severe and progressive retinal degeneration with concomitant loss of retinal function that mimics retinitis pigmentosa due to mutations in the CRB1 gene. Here, we studied the effects of cell-type-specific loss of CRB2 from the developing mouse retina using targeted conditional deletion of Crb2 in photoreceptors or Müller cells. We analyzed the consequences of targeted loss of CRB2 in the adult mouse retina using adeno-associated viral vectors encoding Cre recombinase and short hairpin RNA against Crb2. In vivo retinal imaging by means of optical coherence tomography on retinas lacking CRB2 in photoreceptors showed progressive thinning of the photoreceptor layer and cellular mislocalization. Electroretinogram recordings under scotopic conditions showed severe attenuation of the a-wave, confirming the degeneration of photoreceptors. Retinas lacking CRB2 in developing photoreceptors showed early onset of abnormal lamination, whereas retinas lacking CRB2 in developing Müller cells showed late onset retinal disorganization. Our data suggest that in the developing retina, CRB2 has redundant functions in Müller glial cells, while CRB2 has essential functions in photoreceptors. Our data suggest that short-term loss of CRB2 in adult mouse photoreceptors, but not in Müller glial cells, causes sporadic loss of adhesion between photoreceptors and Müller cells.

Mosaic synaptopathy and functional defects in Cav1.4 heterozygous mice and human carriers of CSNB2.

Mutations in CACNA1F encoding the α1-subunit of the retinal Cav1.4 L-type calcium channel have been linked to Cav1.4 channelopathies including incomplete congenital stationary night blindness type 2A (CSNB2), Åland Island eye disease (AIED) and cone-rod dystrophy type 3 (CORDX3). Since CACNA1F is located on the X chromosome, Cav1.4 channelopathies are typically affecting male patients via X-chromosomal recessive inheritance. Occasionally, clinical symptoms have been observed in female carriers, too. It is currently unknown how these mutations lead to symptoms in carriers and how the retinal network in these females is affected. To investigate these clinically important issues, we compared retinal phenotypes in Cav1.4-deficient and Cav1.4 heterozygous mice and in human female carrier patients. Heterozygous Cacna1f carrier mice have a retinal mosaic consistent with differential X-chromosomal inactivation, characterized by adjacent vertical columns of affected and non-affected wild-type-like retinal network. Vertical columns in heterozygous mice are well comparable to either the wild-type retinal network of normal mice or to the retina of homozygous mice. Affected retinal columns display pronounced rod and cone photoreceptor synaptopathy and cone degeneration. These changes lead to vastly impaired vision-guided navigation under dark and normal light conditions and reduced retinal electroretinography (ERG) responses in Cacna1f carrier mice. Similar abnormal ERG responses were found in five human CACNA1F carriers, four of which had novel mutations. In conclusion, our data on Cav1.4 deficient mice and human female carriers of mutations in CACNA1F are consistent with a phenotype of mosaic CSNB2.

Targeted ablation of CRB1 and CRB2 in retinal progenitor cells mimics Leber congenital amaurosis.

Development in the central nervous system is highly dependent on the regulation of the switch from progenitor cell proliferation to differentiation, but the molecular and cellular events controlling this process remain poorly understood. Here, we report that ablation of Crb1 and Crb2 genes results in severe impairment of retinal function, abnormal lamination and thickening of the retina mimicking human Leber congenital amaurosis due to loss of CRB1 function. We show that the levels of CRB1 and CRB2 proteins are crucial for mouse retinal development, as they restrain the proliferation of retinal progenitor cells. The lack of these apical proteins results in altered cell cycle progression and increased number of mitotic cells leading to an increased number of late-born cell types such as rod photoreceptors, bipolar and Müller glia cells in postmitotic retinas. Loss of CRB1 and CRB2 in the retina results in dysregulation of target genes for the Notch1 and YAP/Hippo signaling pathways and increased levels of P120-catenin. Loss of CRB1 and CRB2 result in altered progenitor cell cycle distribution with a decrease in number of late progenitors in G1 and an increase in S and G2/M phase. These findings suggest that CRB1 and CRB2 suppress late progenitor pool expansion by regulating multiple proliferative signaling pathways.