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

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

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