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The neuronal ceroid lipofuscinoses (NCLs), often referred to as Batten disease, are inherited lysosomal storage disorders that represent the most common neurodegeneration during childhood. Symptoms include seizures, vision loss, motor and cognitive decline, and premature death. The development of brain-directed treatments for NCLs has made noteworthy progress in recent years. Clinical trials are currently ongoing or planned for different forms of the disease. Despite these promising advances, it is unlikely that therapeutic interventions targeting the brain will prevent loss of vision in patients as retinal cells remain untreated and will continue to degenerate. Here, we demonstrate that Cln3(Δex7/8) mice, a mouse model of CLN3 Batten disease with juvenile onset, suffer from a decline in inner retinal function resulting from the death of rod bipolar cells, interneurons vital for signal transmission from photoreceptors to ganglion cells in the retina. We also show that this ocular phenotype can be treated by adeno-associated virus (AAV)-mediated expression of CLN3 in cells of the inner retina, leading to significant survival of bipolar cells and preserved retinal function. In contrast, the treatment of photoreceptors, which are lost in patients at late disease stages, was not therapeutic in Cln3(Δex7/8) mice, underlining the notion that CLN3 disease is primarily a disease of the inner retina with secondary changes in the outer retina. These data indicate that bipolar cells play a central role in this disease and identify this cell type as an important target for ocular AAV-based gene therapies for CLN3 disease.

Opticin is an endogenous vitreous glycoprotein that may have therapeutic potential as it has been shown that supra-normal concentrations supress preretinal neovascularisation. Herein we investigated the pharmacokinetics of opticin following intravitreal injection in rabbits. To measure simultaneously concentrations of human and rabbit opticin, a selected reaction monitoring mass spectrometry assay was developed. The mean concentration of endogenous rabbit opticin in 7 uninjected eyes was measured and found to be 19.2 nM or 0.62 µg/ml. When the vitreous was separated by centrifugation into a supernatant and collagen-containing pellet, 94 % of the rabbit opticin was in the supernatant. Intravitreal injection of human opticin (40 µg) into both eyes of rabbits was followed by enucleation at 5 h, 24 h, 72 h, 7 days, 14 days and 28 days post-injection (n=6 at each time point) and measurement of vitreous human and rabbit opticin concentrations in the supernatant and collagen-containing pellet following centrifugation. The volume of distribution of human opticin was calculated to be 3.31 ml and the vitreous half-life 4.2 days. Assuming that rabbit and human opticin are cleared from rabbit vitreous at the same rate, opticin is secreted into the vitreous at a rate of 0.14 µg/day. We conclude that intravitreally injected opticin has a vitreous half-life that is similar to currently available anti-angiogenic therapeutics. Whilst opticin was first identified bound to vitreous collagen fibrils, here we demonstrate that >90% of endogenous opticin is not bound to collagen. Endogenous opticin is secreted by the non-pigmented ciliary epithelium into the rabbit vitreous at a remarkably high rate and the turnover in vitreous is approximately 15% per day.

The neuronal ceroid lipofuscinoses (NCLs), more commonly referred to as Batten disease, are a group of inherited lysosomal storage disorders that present with neurodegeneration, loss of vision and premature death. There are at least 13 genetically distinct forms of NCL. Enzyme replacement therapies and pre-clinical studies on gene supplementation have shown promising results for NCLs caused by lysosomal enzyme deficiencies. The development of gene therapies targeting the brain for NCLs caused by defects in transmembrane proteins has been more challenging and only limited therapeutic effects in animal models have been achieved so far. Here, we describe the development of an adeno-associated virus (AAV)-mediated gene therapy to treat the neurodegeneration in a mouse model of CLN6 disease, a form of NCL with a deficiency in the membrane-bound protein CLN6. We show that neonatal bilateral intracerebroventricular injections with AAV9 carrying CLN6 increase lifespan by more than 90%, maintain motor skills and motor coordination and reduce neuropathological hallmarks of Cln6-deficient mice up to 23 months post vector administration. These data demonstrate that brain-directed gene therapy is a valid strategy to treat the neurodegeneration of CLN6 disease and may be applied to other forms of NCL caused by transmembrane protein deficiencies in the future.

In the adult central nervous system, endothelial and neuronal cells engage in tight cross-talk as key components of the so-called neurovascular unit. Impairment of their critical relationship adversely affects tissue homeostasis, as observed in neurodegenerative conditions including Alzheimer's and Parkinson's disease. In development, the influence of neuroprogenitor cells on angiogenesis is poorly understood. Here, we show that these cells interact intimately with the growing retinal vascular network, and we identify a novel regulatory mechanism of vasculature development mediated by hypoxia-inducible factor 2a (Hif2a). By Cre-lox gene excision, we show that Hif2a in retinal neuroprogenitor cells upregulates the expression of the pro-angiogenic mediators vascular endothelial growth factor and erythropoietin, whereas it locally downregulates the angiogenesis inhibitor endostatin. Importantly, absence of Hif2a in retinal neuroprogenitor cells causes a marked reduction of proliferating endothelial cells at the angiogenic front. This results in delayed retinal vascular development, fewer major retinal vessels and reduced density of the peripheral deep retinal vascular plexus. Our findings demonstrate that retinal neuroprogenitor cells are a critical component of the developing neurovascular unit.

The neuronal ceroid lipofuscinoses (NCLs) are inherited lysosomal storage disorders characterized by general neurodegeneration and premature death. Sight loss is also a major symptom in NCLs, severely affecting the quality of life of patients, but it is not targeted effectively by brain-directed therapies. Here we set out to explore the therapeutic potential of an ocular gene therapy to treat sight loss in NCL due to a deficiency in the transmembrane protein CLN6. We found that, although Cln6nclf mice presented mainly with photoreceptor degeneration, supplementation of CLN6 in photoreceptors was not beneficial. Because the level of CLN6 is low in photoreceptors but high in bipolar cells (retinal interneurons that are only lost in Cln6-deficient mice at late disease stages), we explored the therapeutic effects of delivering CLN6 to bipolar cells using adeno-associated virus (AAV) serotype 7m8. Bipolar cell-specific expression of CLN6 slowed significantly the loss of photoreceptor function and photoreceptor cells. This study shows that the deficiency of a gene normally expressed in bipolar cells can cause the loss of photoreceptors and that this can be prevented by bipolar cell-directed treatment.