Intraparenchymal convection enhanced delivery of AAV in sheep to treat Mucopolysaccharidosis IIIC.

BACKGROUND: Mucopolysaccharidosis IIIC (MPSIIIC) is one of four Sanfilippo diseases sharing clinical symptoms of severe cognitive decline and shortened lifespan. The missing enzyme, heparan sulfate acetyl-CoA: α-glucosaminide-N-acetyltransferase (HGSNAT), is bound to the lysosomal membrane, therefore cannot cross the blood-brain barrier or diffuse between cells. We previously demonstrated disease correction in MPSIIIC mice using an Adeno-Associated Vector (AAV) delivering HGSNAT via intraparenchymal brain injections using an AAV2 derived AAV-truetype (AAV-TT) serotype with improved distribution over AAV9. METHODS: Here, intraparenchymal AAV was delivered in sheep using catheters or Hamilton syringes, placed using Brainlab cranial navigation for convection enhanced delivery, to reduce proximal vector expression and improve spread. RESULTS: Hamilton syringes gave improved AAV-GFP distribution, despite lower vector doses and titres. AAV-TT-GFP displayed moderately better transduction compared to AAV9-GFP but both serotypes almost exclusively transduced neurons. Functional HGSNAT enzyme was detected in 24-37% of a 140g gyrencephalic sheep brain using AAV9-HGSNAT with three injections in one hemisphere. CONCLUSIONS: Despite variabilities in volume and titre, catheter design may be critical for efficient brain delivery. These data help inform a clinical trial for MPSIIIC.

Retinopathy in Greyhound dogs: Prevalence, fundoscopic, and histopathological findings.

OBJECTIVES: To determine the prevalence of retinal lesions and describe the fundoscopic findings of retinopathy in Greyhound dogs in the Manawatu/Whanganui region of New Zealand. To examine possible associations between sex, age, and racing variables with retinopathy in the study population. To describe retinal histologic findings in seven Greyhounds with retinopathy in New Zealand. METHODS: Two hundred Greyhound dogs from the Manawatu/Whanganui region of New Zealand underwent fundoscopy and fundic photography to identify and score the degree of retinopathy. Associations between retinopathy and age, sex, as well as racing variables, were examined. Histologic examination of the retina was undertaken on the eyes of seven Greyhounds from the Manawatu and Canterbury regions previously diagnosed with retinopathy by fundoscopy. RESULTS: Fifty dogs (25.1%) were identified with retinopathy of varying degrees of severity. In at least one eye, 7.5% of dogs had mild retinopathy, 11.6% moderate retinopathy, and 6.0% severe retinopathy. Males were more likely to be affected in both eyes and with moderate or severe grades, than females. Increasing age was not associated with increased prevalence of retinopathy, nor increased grade of severity. Retinal histology identified multifocal retinal detachment in 5 of the 7 cases examined and other common lesions included choroidal necrosis and outer to full-thickness retinal atrophy in the absence of significant inflammation. CONCLUSIONS: Retinopathy is prevalent in Greyhounds in the Manawatu/Whanganui region of New Zealand, but more research is required to elucidate the etiopathogenesis. Consideration should be made to include mandatory eye health examination in racing Greyhound dogs.

Intravitreal gene therapy protects against retinal dysfunction and degeneration in sheep with CLN5 Batten disease.

Neuronal ceroid lipofuscinoses (NCL; Batten disease) are a group of inherited neurodegenerative diseases primarily affecting children. A common feature across most NCLs is the progressive loss of vision. We performed intravitreal injections of self-complementary AAV9 vectors packaged with either ovine CLN5 or CLN6 into one eye of 3-month-old CLN5-/- or CLN6-/- animals, respectively. Electroretinography (ERG) was performed every month following treatment, and retinal histology was assessed post-mortem in the treated compared to untreated eye. In CLN5-/- animals, ERG amplitudes were normalised in the treated eye whilst the untreated eye declined in a similar manner to CLN5 affected controls. In CLN6-/- animals, ERG amplitudes in both eyes declined over time although the treated eye showed a slower decline. Post-mortem examination revealed significant attenuation of retinal atrophy and lysosomal storage body accumulation in the treated eye compared with the untreated eye in CLN5-/- animals. This proof-of-concept study provides the first observation of efficacious intravitreal gene therapy in a large animal model of NCL. In particular, the single administration of AAV9-mediated intravitreal gene therapy can successfully ameliorate retinal deficits in CLN5-/- sheep. Combining ocular gene therapy with brain-directed therapy presents a promising treatment strategy to be used in future sheep trials aiming to halt neurological and retinal disease in CLN5 Batten disease.

Longitudinal In Vivo Monitoring of the CNS Demonstrates the Efficacy of Gene Therapy in a Sheep Model of CLN5 Batten Disease.

Neuronal ceroid lipofuscinoses (NCLs; Batten disease) are neurodegenerative lysosomal storage diseases predominantly affecting children. Single administration of brain-directed lentiviral or recombinant single-stranded adeno-associated virus 9 (ssAAV9) vectors expressing ovine CLN5 into six pre-clinically affected sheep with a naturally occurring CLN5 NCL resulted in long-term disease attenuation. Treatment efficacy was demonstrated by non-invasive longitudinal in vivo monitoring developed to align with assessments used in human medicine. The treated sheep retained neurological and cognitive function, and one ssAAV9-treated animal has been retained and is now 57 months old, almost triple the lifespan of untreated CLN5-affected sheep. The onset of visual deficits was much delayed. Computed tomography and MRI showed that brain structures and volumes remained stable. Because gene therapy in humans is more likely to begin after clinical diagnosis, self-complementary AAV9-CLN5 was injected into the brain ventricles of four 7-month-old affected sheep already showing early clinical signs in a second trial. This also halted disease progression beyond their natural lifespan. These findings demonstrate the efficacy of CLN5 gene therapy, using three different vector platforms, in a large animal model and, thus, the prognosis for human translation.

An EEG Investigation of Sleep Homeostasis in Healthy and CLN5 Batten Disease Affected Sheep.

UNLABELLED: Sheep have large brains with human-like anatomy, making them a useful species for studying brain function. Sleep homeostasis has not been studied in sheep. Here, we establish correlates of sleep homeostasis in sheep through a sleep deprivation experiment. We then use these correlates to elucidate the nature of sleep deficits in a naturally occurring ovine model of neuronal ceroid lipofuscinosis (NCL, Batten disease) caused by a mutation in CLN5 In humans, mutations in this gene lead to cortical atrophy and blindness, as well as sleep abnormalities. We recorded electroencephalograms (EEGs) from unaffected and early stage CLN5(-/-) (homozygous, affected) sheep over 3 consecutive days, the second day being the sleep deprivation day. In unaffected sheep, sleep deprivation led to increased EEG delta (0.5-4 Hz) power during non-rapid eye movement (NREM) sleep, increased time spent in the NREM sleep state, and increased NREM sleep bout length. CLN5(-/-) sheep showed comparable increases in time spent in NREM sleep and NREM sleep bout duration, verifying the presence of increased sleep pressure in both groups. Importantly, CLN5(-/-) sheep did not show the increase in NREM sleep delta power seen in unaffected sheep. This divergent delta power response is consistent with the known cortical degeneration in CLN5(-/-) sheep. We conclude that, whereas sleep homeostasis is present in CLN5(-/-) sheep, underlying CLN5(-/-) disease processes prevent its full expression, even at early stages. Such deficits may contribute to early abnormalities seen in sheep and patients and warrant further study. SIGNIFICANCE STATEMENT: Sleep abnormalities pervade most neurological diseases, including the neuronal ceroid lipofuscinoses (NCLs). Here, we show that, in an ovine model of a variant late-infantile NCL, there is abnormal expression of sleep homeostasis. Whereas some sleep pressure correlates respond to sleep deprivation, the strongest electroencephalogram (EEG) correlate of sleep pressure, non-REM delta power, failed to increase. This highlights the relevance of sleep deficits in this disease, in which the drive for sleep exists but the underlying disease prevents its full expression. Sleep abnormalities could contribute to early disease symptoms such as behavioral disorder and cognitive decline. Our study also shows sleep homeostatic EEG correlates in sheep, opening up new opportunities for studying sleep in a large social mammal with complex human-like brain neuroanatomy.

Characterisation of early changes in ovine CLN5 and CLN6 Batten disease neural cultures for the rapid screening of therapeutics.

Batten disease (neuronal ceroid lipofuscinosis) refers to a group of neurodegenerative lysosomal storage diseases predominantly affecting children. There are currently no effective treatments, and the functions of many of the associated gene products are unknown. Here we characterise fetal neural cultures from two genetically distinct sheep forms of Batten disease, with mutations in the lysosomal protein encoding gene CLN5 and endoplasmic reticulum membrane protein encoding gene CLN6, respectively. We found similar reductions in autophagy, acidic organelles and synaptic recycling in both forms compared to unaffected cells. We then developed a high-throughput screen and tested for correction of deficient cells with lentiviral-mediated CLN5 or CLN6 gene transfer and fibrate drugs, gemfibrozil and fenofibrate in CLN6 deficient neural cultures. These assays provide a simple system to rapidly screen candidate therapies or libraries of drugs prior to in vivo testing.

Recent studies of ovine neuronal ceroid lipofuscinoses from BARN, the Batten Animal Research Network.

Studies on naturally occurring New Zealand and Australian ovine models of the neuronal ceroid-lipofuscinoses (Batten disease, NCLs) have greatly aided our understanding of these diseases. Close collaborations between the New Zealand groups at Lincoln University and the University of Otago, Dunedin, and a group at the University of Sydney, Australia, led to the formation of BARN, the Batten Animal Research Network. This review focusses on presentations at the 14th International Conference on Neuronal Ceroid Lipofuscinoses (Batten Disease), recent relevant background work, and previews of work in preparation for publication. Themes include CLN5 and CLN6 neuronal cell culture studies, studies on tissues from affected and control animals and whole animal in vivo studies. Topics include the effect of a CLN6 mutation on endoplasmic reticulum proteins, lysosomal function and the interactions of CLN6 with other lysosomal activities and trafficking, scoping gene-based therapies, a molecular dissection of neuroinflammation, identification of differentially expressed genes in brain tissue, an attempted therapy with an anti-inflammatory drug in vivo and work towards gene therapy in ovine models of the NCLs. This article is part of a Special Issue entitled: "Current Research on the Neuronal Ceroid Lipofuscinoses (Batten Disease)".

Translational neurophysiology in sheep: measuring sleep and neurological dysfunction in CLN5 Batten disease affected sheep.

Creating valid mouse models of slowly progressing human neurological diseases is challenging, not least because the short lifespan of rodents confounds realistic modelling of disease time course. With their large brains and long lives, sheep offer significant advantages for translational studies of human disease. Here we used normal and CLN5 Batten disease affected sheep to demonstrate the use of the species for studying neurological function in a model of human disease. We show that electroencephalography can be used in sheep, and that longitudinal recordings spanning many months are possible. This is the first time such an electroencephalography study has been performed in sheep. We characterized sleep in sheep, quantifying characteristic vigilance states and neurophysiological hallmarks such as sleep spindles. Mild sleep abnormalities and abnormal epileptiform waveforms were found in the electroencephalographies of Batten disease affected sheep. These abnormalities resemble the epileptiform activity seen in children with Batten disease and demonstrate the translational relevance of both the technique and the model. Given that both spontaneous and engineered sheep models of human neurodegenerative diseases already exist, sheep constitute a powerful species in which longitudinal in vivo studies can be conducted. This will advance our understanding of normal brain function and improve our capacity for translational research into neurological disorders.

Inhibition of storage pathology in prenatal CLN5-deficient sheep neural cultures by lentiviral gene therapy.

The neuronal ceroid lipofuscinoses (NCLs, Batten disease) are inherited neurodegenerative lysosomal storage diseases caused by mutations in several different genes. Mutations in CLN5 cause a variant late-infantile human disease and some cases of juvenile and adult clinical disease. NCLs also occur in animals, and a flock of New Zealand Borderdale sheep with a CLN5 splice-site mutation has been developed for model studies. Dissociated mixed neural cells from CLN5-deficient foetal sheep brains contained no obvious storage bodies at plating but these accumulated rapidly in culture, mainly in microglial cells and also in neurons and astrocytes. Accumulation was very obvious after a week, as monitored by fluorescent microscopy and immunostaining for subunit c of mitochondrial ATP synthase. Photography at intervals revealed the dynamic nature of the cultures and a flow of storage bodies between cells, specifically the phagocytosis of storage-body containing cells by microglia and incorporation of the storage bodies into the host cells. No storage was observed in cultured control cells. Transduction of cell cultures with a lentiviral vector expressing a C-terminal Myc tagged CLN5 resulted in secretion of post-translationally glycosylated and processed CLN5. Transduction of CLN5-deficient cultures with this construct rapidly reversed storage body accumulation, to less than half in only six days. These results show that storage body accumulation is reversible with enzyme correction and support the use of these cultures for testing of therapeutics prior to whole animal studies.

Characterization of neuropathology in ovine CLN5 and CLN6 neuronal ceroid lipofuscinoses (Batten disease).

Sheep with naturally occurring CLN5 and CLN6 forms of neuronal ceroid lipofuscinoses (Batten disease) share the key clinical features of the human disease and represent an ideal model system in which the clinical efficacy of gene therapies is developed and test. However, it was first important to characterize the neuropathological changes that occur with disease progression in affected sheep. This study compared neurodegeneration, neuroinflammation, and lysosomal storage accumulation in CLN5 affected Borderdale, CLN6 affected South Hampshire, and Merino sheep brains from birth to end-stage disease at ≤24 months of age. Despite very different gene products, mutations, and subcellular localizations, the pathogenic cascade was remarkably similar for all three disease models. Glial activation was present at birth in affected sheep and preceded neuronal loss, with both spreading from the visual and parieto-occipital cortices most prominently associated with clinical symptoms to the entire cortical mantle by end-stage disease. In contrast, the subcortical regions were less involved, yet lysosomal storage followed a near-linear increase across the diseased sheep brain with age. Correlation of these neuropathological changes with published clinical data identified three potential therapeutic windows in affected sheep-presymptomatic (3 months), early symptomatic (6 months), and a later symptomatic disease stage (9 months of age)-beyond which the extensive depletion of neurons was likely to diminish any chance of therapeutic benefit. This comprehensive natural history of the neuropathological changes in ovine CLN5 and CLN6 disease will be integral in determining what impact treatment has at each of these disease stages.

Long-term safety and dose escalation of intracerebroventricular CLN5 gene therapy in sheep supports clinical translation for CLN5 Batten disease.

CLN5 neuronal ceroid lipofuscinosis (NCL, Batten disease) is a rare, inherited fatal neurodegenerative disorder caused by mutations in the CLN5 gene. The disease is characterised by progressive neuronal loss, blindness, and premature death. There is no cure. This study evaluated the efficacy of intracerebroventricular (ICV) delivery of an adeno-associated viral vector encoding ovine CLN5 (scAAV9/oCLN5) in a naturally occurring sheep model of CLN5 disease. CLN5 affected (CLN5-/-) sheep received low, moderate, or high doses of scAAV9/oCLN5 at three disease stages. The treatment delayed disease progression, extended survival and slowed stereotypical brain atrophy in most animals. Of note, one high dose treated animal only developed mild disease symptomology and survived to 60.1 months of age, triple the natural life expectancy of an untreated CLN5-/- sheep. Eyesight was not preserved at any administration age or dosage. Histopathologic examination revealed that greater transduction efficiency was achieved through higher ICV doses, and this resulted in greater amelioration of disease pathology. Together with other pre-clinical data from CLN5-/- sheep, the safety and efficacy data from these investigational new drug (IND)-enabling studies supported the initiation of the first in-human CLN5 gene therapy clinical study using the ICV delivery route for the treatment of CLN5 NCL. Clinical Trial Registration: https://clinicaltrials.gov/, identifier NCT05228145.

Progressive MRI brain volume changes in ovine models of CLN5 and CLN6 neuronal ceroid lipofuscinosis.

Neuronal ceroid lipofuscinoses (Batten disease) are a group of inherited lysosomal storage disorders characterized by progressive neurodegeneration leading to motor and cognitive dysfunction, seizure activity and blindness. The disease can be caused by mutations in 1 of 13 ceroid lipofuscinosis neuronal (CLN) genes. Naturally occurring sheep models of the CLN5 and CLN6 neuronal ceroid lipofuscinoses recapitulate the clinical disease progression and post-mortem pathology of the human disease. We used longitudinal MRI to assess global and regional brain volume changes in CLN5 and CLN6 affected sheep compared to age-matched controls over 18 months. In both models, grey matter volume progressively decreased over time, while cerebrospinal fluid volume increased in affected sheep compared with controls. Total grey matter volume showed a strong positive correlation with clinical scores, while cerebrospinal fluid volume was negatively correlated with clinical scores. Cortical regions in affected animals showed significant atrophy at baseline (5 months of age) and progressively declined over the disease course. Subcortical regions were relatively spared with the exception of the caudate nucleus in CLN5 affected animals that degenerated rapidly at end-stage disease. Our results, which indicate selective vulnerability and provide a timeline of degeneration of specific brain regions in two sheep models of neuronal ceroid lipofuscinoses, will provide a clinically relevant benchmark for assessing therapeutic efficacy in subsequent trials of gene therapy for CLN5 and CLN6 disease.

Efficacy of dual intracerebroventricular and intravitreal CLN5 gene therapy in sheep prompts the first clinical trial to treat CLN5 Batten disease.

Mutations in the CLN5 gene cause the fatal, pediatric, neurodegenerative disease CLN5 neuronal ceroid lipofuscinosis. Affected children suffer progressive neuronal loss, visual failure and premature death. Presently there is no treatment. This study evaluated dual intracerebroventricular (ICV) and intravitreal (IVT) administration of a self-complementary adeno-associated viral vector encoding ovine CLN5 (scAAV9/oCLN5) into CLN5 affected sheep (CLN5-/-) at various disease stages. CLN5 disease progression was slowed in pre-symptomatic sheep who received a moderate dose of scAAV9/oCLN5, whilst a higher ICV dose treatment in early and advanced symptomatic animals delayed or halted disease progression. Intracranial (brain) volume loss was attenuated in all treatment cohorts, and visual function was also sustained in both the early and advanced symptomatic treated sheep over the 24-month duration of the study. Robust CLN5 protein expression was detected throughout the brain and spinal cord, and improvements in central nervous system and retinal disease correlates were observed. These findings hold translational promise for extending and improving the quality of life in both pre-symptomatic and symptomatic CLN5 patients, and prompted the initiation of the first in-human Phase I/II clinical trial testing ICV/IVT administration of scAAV9 encoding human CLN5 (https://clinicaltrials.gov/; NCT05228145).

Intravitreal Injections in the Ovine Eye.

There are several methods for the delivery of therapeutic agents to the retina, including intravitreal (IVT), subretinal, suprachoroidal, periocular, or topical administration. IVT drug delivery involves an injection into the vitreous humor of the eye, a gelatinous substance that fills the posterior chamber of the eye and maintains the shape of the eye globe. Although the IVT route is less specifically targeted than subretinal delivery, it is much less invasive and is widely used in clinical settings for a range of ocular diseases. We previously demonstrated the efficacy of intravitreal delivery of an adeno-associated virus (AAV)-mediated gene therapy product (AAV9.CLN5) in sheep with a naturally occurring CLN5 form of neuronal ceroid lipofuscinosis (NCL). Affected sheep received IVT gene therapy in one eye, with the other untreated eye serving as an internal control. Retinal structure and function were maintained in the treated eye up to 15 months after treatment, while the untreated eye displayed progressively declining function and severe atrophy during postmortem examination. Based on the sheep studies, the CLN5 gene therapy product was cleared as a candidate investigational new drug (IND) by the United States Food and Drug Administration in September 2021. This paper details the surgical protocol for IVT delivery of a therapeutic viral vector to the ovine eye.

The Translational Benefits of Sheep as Large Animal Models of Human Neurological Disorders.

The past two decades have seen a considerable rise in the use of sheep to model human neurological disorders. While each animal model has its merits, sheep have many advantages over small animal models when it comes to studies on the brain. In particular, sheep have brains more comparable in size and structure to the human brain. They also have much longer life spans and are docile animals, making them useful for a wide range of in vivo studies. Sheep are amenable to regular blood and cerebrospinal fluid sampling which aids in biomarker discovery and monitoring of treatment efficacy. Several neurological diseases have been found to occur naturally in sheep, however sheep can also be genetically engineered or experimentally manipulated to recapitulate disease or injury. Many of these types of sheep models are currently being used for pre-clinical therapeutic trials, particularly gene therapy, with studies from several models culminating in potential treatments moving into clinical trials. This review will provide an overview of the benefits of using sheep to model neurological conditions, and highlight naturally occurring and experimentally induced sheep models that have demonstrated translational validity.

Modelling Neurological Diseases in Large Animals: Criteria for Model Selection and Clinical Assessment.

Issue: The impact of neurological disorders is recognised globally, with one in six people affected in their lifetime and few treatments to slow or halt disease progression. This is due in part to the increasing ageing population, and is confounded by the high failure rate of translation from rodent-derived therapeutics to clinically effective human neurological interventions. Improved translation is demonstrated using higher order mammals with more complex/comparable neuroanatomy. These animals effectually span this translational disparity and increase confidence in factors including routes of administration/dosing and ability to scale, such that potential therapeutics will have successful outcomes when moving to patients. Coupled with advancements in genetic engineering to produce genetically tailored models, livestock are increasingly being used to bridge this translational gap. Approach: In order to aid in standardising characterisation of such models, we provide comprehensive neurological assessment protocols designed to inform on neuroanatomical dysfunction and/or lesion(s) for large animal species. We also describe the applicability of these exams in different large animals to help provide a better understanding of the practicalities of cross species neurological disease modelling. Recommendation: We would encourage the use of these assessments as a reference framework to help standardise neurological clinical scoring of large animal models.

Electroretinography data from ovine models of CLN5 and CLN6 neuronal ceroid lipofuscinoses.

This article presents datasets associated with the research article entitled "Intravitreal gene therapy protects against retinal dysfunction and degeneration in sheep with CLN5 Batten disease" (Murray et al., [1]). The neuronal ceroid lipofuscinoses (NCL; Batten disease) are a group of fatal inherited diseases caused by mutations in a number of CLN genes that lead to degenerative and fatal encephalopathies in children. Naturally-occuring sheep models of NCL exist. Affected sheep share the clinical and pathological features of the human disease, including retinal degeneration. Electroretinography (ERG) was employed to characterise the physiological changes in the degenerating retina of CLN5 and CLN6 forms of ovine NCL. ERGs were performed every two months from 3 to 17 months of age in 11 NCL affected (6 CLN5-/ - and 5 CLN6-/- ) sheep and 12 clinically normal heterozygous controls (6 CLN5+/ - and 6 CLN6 +/-) under three different adaptation conditions. A-wave and b-wave amplitudes were collected from each eye using the Eickemeyer Veterinary ERG system. These are the first longitudinal datasets assessing the progression of retinal degeneration in ovine NCL, aiding in characterisation of the disease process and providing insight into optimal therapeutic windows for subsequent studies.

Practical implications of motion correction with motion insensitive radial k-space acquisitions in MRI.

OBJECTIVE: To highlight specific instances when radial k-space acquisitions in MRI result in image artifacts and how to ameliorate such artifacts. METHODS: We acquired axial T2 weighted MR images on (1) the American College of Radiology (ACR) phantom and (2) a sedated sheep with rectilinear and multiblade radial k-space filling acquisitions. Images were acquired on four (2 × 1.5T and 2 × 3T) different MRI scanners. For the radial k-space acquisitions, we acquired images with and without motion correction. All images were visually inspected for the presence of artifact. RESULTS: Images collected via the conventional rectilinear method were of diagnostic quality and free of artifact. Both ACR and sheep images acquired with radial k-space acquisitions and motion correction suffered significant artifact at different slice locations, scan sessions and across all the four scanners. Severity of the artifact was associated with echo train length. However, the artifact was eliminated when motion correction was not employed. CONCLUSION: When little to no motion is present, the use of motion correction with radial k-space acquisitions can compromise image quality. However, image quality is quickly improved, and the artifact eliminated, by repeating the scan without motion correction or by using a conventional rectilinear alternative. Advances in Knowledge: By improving awareness and understanding of this artifact, MRI users will be able to adjust MRI protocols, resulting in more successful scanning sessions, better image quality, fewer call backs and increased diagnostic confidence.

Computed tomography provides enhanced techniques for longitudinal monitoring of progressive intracranial volume loss associated with regional neurodegeneration in ovine neuronal ceroid lipofuscinoses.

INTRODUCTION: The neuronal ceroid lipofuscinoses (NCLs; Batten disease) are a group of fatal neurodegenerative lysosomal storage diseases of children caused by various mutations in a range of genes. Forms associated with mutations in two of these, CLN5 and CLN6, are being investigated in well-established sheep models. Brain atrophy leading to psychomotor degeneration is among the defining features, as is regional progressive ossification of the inner cranium. Ongoing viral-mediated gene therapy trials in these sheep are yielding encouraging results. In vivo assessment of brain atrophy is integral to the longitudinal monitoring of individual animals and provides robust data for translation to treatments for humans. METHODS: Computed tomography (CT)-based three-dimensional reconstruction of the intracranial volume (ICV) over time reflects the progression of cortical brain atrophy, verifying the use of ICV measurements as a surrogate measure for brain size in ovine NCL. RESULTS: ICVs of NCL-affected sheep increase for the first few months, but then decline progressively between 5 and 13 months in CLN5-/- sheep and 11-15 months in CLN6-/- sheep. Cerebral ventricular volumes are also increased in affected animals. To facilitate ICV measures, the radiodensities of ovine brain tissue and cerebrospinal fluid were identified. Ovine brain tissue exhibited a Hounsfield unit (HU) range of (24; 56) and cerebrospinal fluid a HU range of (-12; 23). CONCLUSIONS: Computed tomography scanning and reconstruction verify that brain atrophy ovine CLN5 NCL originates in the occipital lobes with subsequent propagation throughout the whole cortex and these regional differences are reflected in the ICV loss.

Neuronal ceroid lipofuscinosis in Devon cattle is caused by a single base duplication (c.662dupG) in the bovine CLN5 gene.

The neuronal ceroid lipofuscinoses (NCLs, Batten disease) are recessively inherited neurodegenerative disorders that affect humans and other animals, characterised by brain atrophy and the accumulation of lysosome derived fluorescent storage bodies in neurons and most other cells. Common clinical signs include blindness, ataxia, dementia, seizures and premature death. The associated genes for six different human forms have been identified (CLN1, CLN2, CLN3, CLN5, CLN6 and CLN8), and three other human forms suggested (CLNs 4, 7 and 9). A form of NCL in Australian Devon cattle is caused by a single base duplication (c.662dupG) in bovine CLN5. This mutation causes a frame-shift and premature termination (p.Arg221GlyfsX6) which is predicted to result in a severely truncated protein, analogous to disease causing mutations in human Finnish late infantile variant NCL (CLN5), and a simple genetic diagnostic test has been developed. The symptoms and disease course in cattle also matches CLN5. Only one initiation site was found in the bovine gene, equivalent to the third of four possible initiation sites in the human gene. As cattle are anatomically and physiologically similar to humans with a human-like central nervous system and easy to maintain and breed, they provide a valuable alternative model for CLN5 studies.