Gene therapy restores vision in rd1 mice after removal of a confounding mutation in Gpr179

The rd1 mouse with a mutation in the Pde6b gene was the first strain of mice identified with a retinal degeneration. However, AAV-mediated gene supplementation of rd1 mice only results in structural preservation of photoreceptors, and restoration of the photoreceptor-mediated a-wave, but not in restoration of the bipolar cell-mediated b-wave. Here we show that a mutation in Gpr179 prevents the full restoration of vision in rd1 mice. Backcrossing rd1 with C57BL6 mice reveals the complete lack of b-wave in a subset of mice, consistent with an autosomal recessive Mendelian inheritance pattern. We identify a mutation in the Gpr179 gene, which encodes for a G-protein coupled receptor localized to the dendrites of ON-bipolar cells. Gene replacement in rd1 mice that are devoid of the mutation in Gpr179 successfully restores the function of both photoreceptors and bipolar cells, which is maintained for up to 13 months. Our discovery may explain the failure of previous gene therapy attempts in rd1 mice, and we propose that Grp179 mutation status should be taken into account in future studies involving rd1 mice

Multimodal analysis of ocular inflammation using the endotoxin-induced uveitis mouse model

Endotoxin-induced uveitis (EIU) in rodents is a model of acute Toll-like receptor 4 (TLR4)-mediated organ inflammation, and has been used to model human anterior uveitis, examine leukocyte trafficking and test novel anti-inflammatory therapeutics. Wider adoption has been limited by the requirement for manual, non-specific, cell-count scoring of histological sections from each eye as a measure of disease severity. Here, we describe a comprehensive and efficient technique that uses ocular dissection and multimodal tissue analysis. This allows matched disease scoring by multicolour flow cytometric analysis of the inflammatory infiltrate, protein analysis on ocular supernatants and qPCR on remnant tissues of the same eye. Dynamic changes in cell populations could be identified and mapped to chemokine and cytokine changes over the course of the model. To validate the technique, dose-responsive suppression of leukocyte infiltration by recombinant interleukin-10 was demonstrated, as well as selective suppression of the monocyte (CD11b+Ly6C+) infiltrate, in mice deficient for eitherCcl2orCcr2 Optical coherence tomography (OCT) was used for the first time in this model to allowin vivoimaging of infiltrating vitreous cells, and correlated with CD11b+Ly6G+ counts to provide another unique measure of cell populations in the ocular tissue. Multimodal tissue analysis of EIU is proposed as a new standard to improve and broaden the application of this model

Juvenile Batten disease (CLN3): Detailed Ocular Phenotype, Novel Observations, Delayed Diagnosis, Masquerades, and Prospects for Therapy

PURPOSE To characterize the retinal phenotype of juvenile neuronal ceroid lipofuscinosis (JNCL), highlight delayed and mistaken diagnosis, and propose an algorithm for early identification. DESIGN Retrospective case series. SUBJECTS Eight children (5 females) with JNCL. METHODS Review of clinical notes, retinal imaging including fundus autofluorescence (FAF) and optical coherence tomography (OCT), electroretinography (ERG), and both microscopy and molecular genetic testing. MAIN OUTCOME MEASUREMENTS Demographic data, signs and symptoms, visual acuity, FAF and OCT findings, ERG phenotype, and microscopy/molecular genetics. RESULTS Subjects presented with rapid bilateral vision loss over one to eighteen months, with mean visual acuity deteriorating from 0.44 LogMAR (range: 0.20 - 1.78 LogMAR) at baseline, to 1.34 LogMAR (0.30 LogMAR - light perception) at last follow-up. Age of onset ranged from 3 to 7 years (mean 5.3 years). The age at diagnosis of JNCL ranged from 7 to 10 years (mean 8.3 years). Six children displayed eccentric fixation, and six had cognitive or neurological signs at time of diagnosis (75%). Seven patients had bilateral bull’s-eye maculopathy at presentation. Coats-like exudative vasculopathy, not previously reported in JNCL, was observed in one patient. OCT imaging revealed near complete loss of outer retinal layers, and marked atrophy of the nerve fibre and ganglion cell layers, at the central macula. An ‘electronegative’ ERG was present in four patients (50%), but with additional a-wave reduction; there was an undetectable ERG in the remaining four. Blood film microscopy revealed vacuolated lymphocytes and electron microscopy showed lysosomal (fingerprint) inclusions, in all eight patients. CONCLUSIONS In a young child with bilateral rapidly progressive vision loss and macular disturbance, blood film microscopy to detect vacuolated lymphocytes is a rapid, readily accessible, and sensitive screening test for JNCL. Early suspicion of JNCL can be aided by detailed directed history and high-resolution retinal imaging, with subsequent targeted microscopy/genetic testing. Early diagnosis is critical to ensure appropriate management, counselling, support and social care for children and their families. Furthermore, although potential therapies for this group of disorders are in early phase clinical trial, realistic expectations are that successful intervention will be most effective when initiated at the earliest stage of disease

Clinical challenges and future therapeutic approaches for neuronal ceroid lipofuscinosis

© 2019 Elsevier Ltd Treatment of the neuronal ceroid lipofuscinoses, also known as Batten disease, is at the start of a new era because of diagnostic and therapeutic advances relevant to this group of inherited neurodegenerative and life-limiting disorders that affect children. Diagnosis has improved with the use of comprehensive DNA-based tests that simultaneously screen for many genes. The identification of disease-causing mutations in 13 genes provides a basis for understanding the molecular mechanisms underlying neuronal ceroid lipofuscinoses, and for the development of targeted therapies. These targeted therapies include enzyme replacement therapies, gene therapies targeting the brain and the eye, cell therapies, and pharmacological drugs that could modulate defective molecular pathways. Such therapeutic developments have the potential to enable earlier diagnosis and better targeted therapeutic management. The first approved treatment is an intracerebroventricularly administered enzyme for neuronal ceroid lipofuscinosis type 2 disease that delays symptom progression. Efforts are underway to make similar progress for other forms of the disorder

The severity of retinal pathology in homozygous Crb1rd8/rd8 mice is dependent on additional genetic factors.

Understanding phenotype-genotype correlations in retinal degeneration is a major challenge. Mutations in CRB1 lead to a spectrum of autosomal recessive retinal dystrophies with variable phenotypes suggesting the influence of modifying factors. To establish the contribution of the genetic background to phenotypic variability associated with the Crb1(rd8/rd8) mutation, we compared the retinal pathology of Crb1(rd8/rd8)/J inbred mice with that of two Crb1(rd8/rd8) lines backcrossed with C57BL/6JOlaHsd mice. Topical endoscopic fundal imaging and scanning laser ophthalmoscopy fundus images of all three Crb1(rd8/rd8) lines showed a significant increase in the number of inferior retinal lesions that was strikingly variable between the lines. Optical coherence tomography, semithin, ultrastructural morphology and assessment of inflammatory and vascular marker by immunohistochemistry and quantitative reverse transcriptase-polymerase chain reaction revealed that the lesions were associated with photoreceptor death, Müller and microglia activation and telangiectasia-like vascular remodelling-features that were stable in the inbred, variable in the second, but virtually absent in the third Crb1(rd8/rd8) line, even at 12 months of age. This suggests that the Crb1(rd8/rd8) mutation is necessary, but not sufficient for the development of these degenerative features. By whole-genome SNP analysis of the genotype-phenotype correlation, a candidate region on chromosome 15 was identified. This may carry one or more genetic modifiers for the manifestation of the retinal pathology associated with mutations in Crb1. This study also provides insight into the nature of the retinal vascular lesions that likely represent a clinical correlate for the formation of retinal telangiectasia or Coats-like vasculopathy in patients with CRB1 mutations that are thought to depend on such genetic modifiers

Gene therapy to improve vision in neuronal ceroid lipofuscinoses

The neuronal ceroid lipofuscinoses (NCLs) are inherited lysosomal storage disorders that present with severe neurodegeneration and loss of vision. A major obstacle to developing gene therapies for the NCLs is the challenge to efficiently deliver agents throughout the brain; particularly for NCL forms arising from membrane bound protein defects. Adeno-associated virus (AAV) mediated gene therapies have been used for several monogenic retinal degenerations to restore the expression of proteins and improve retinal morphology and function. As vision loss is a key feature in NCL, this thesis sought to explore the therapeutic potential of an ocular AAV-mediated gene therapy in the Cln6nclf mouse, a model deficient in the transmembrane protein Cln6. The ultimate goal of this work is to improve quality of life for patients by developing a therapy to preserve vision. In addition, this study may also help to overcome challenges associated with brain-directed treatments for NCL. To identify the cell population that needs to be therapeutically targeted, we performed an analysis of CLN6 expression in the eye. These data revealed that CLN6 is expressed in photoreceptor and bipolar cells of the retina and that the expression level of CLN6 is higher in bipolar cells than in photoreceptors. We also investigated the retinal phenotype in Cln6nclf mice to determine the time window for treatment and established measures assessing the effects of the treatment on the disease progression. Loss of photoreceptor cells and photoreceptor function occurred as early as 2 and 3 weeks of age ultimately resulting in dramatic thinning of the outer nuclear layer. To test whether we can slow degeneration by gene therapy, we performed subretinal injections targeting photoreceptors in Cln6-deficient mice using AAV2/8.CLN6 vectors. This work demonstrated that despite widespread transgene expression the treatment does not have a beneficial effect on retinal function or morphology. We concluded that photoreceptor treatment was not sufficient and hypothesised that bipolar cells need to be treated additionally to prevent vision loss in Cln6nclf mice. Finally, we investigated strategies to enhance the transduction efficiency of bipolar cells that are poorly transduced by commonly used AAVs. Injections of a recently engineered AAV vector showed widespread transduction of bipolar cells. Currently, we are assessing whether a gene supplementation therapy targeting bipolar cells and photoreceptors is therapeutic in Cln6-deficient mice