Borderdale sheep were genetically diagnosed at birth as either clinically healthy CLN5 heterozygous (CLN5^+/−) or CLN5 affected (CLN5^−/−) as previously described (Frugier et al., 2008). They were maintained at Lincoln University under NIH guidelines for the Care and Use of Animals in Research and the NZ Animal Welfare Act (1999). The animal studies were reviewed and approved by the Lincoln University Animal Ethics and Institutional Biosafety Committees.

Supplementary Table S1 summarizes the study design. Briefly, nine CLN5^-/- ewes received combination bilateral intracerebroventricular (ICV) and unilateral intravitreal (IVT) administration of scAAV9/oCLN5 at three different disease stages: pre-symptomatic 3 months (n=3), early symptomatic 6 months (n=3), or advanced symptomatic 9 months (n=3).

Concurrent age-matched CLN5^+/− (n = 3) and CLN5^−/− (n = 4) ewes acted as untreated clinically normal and affected controls, respectively. Data from these animals were pooled for the neurological examinations and magnetic resonance imaging (MRI) analyses; historic controls were used for the remainder of the in-life assessments and neuropathological analyses.

scAAV9/oCLN5 is a recombinant, self-complementary adeno-associated virus serotype 9 (AAV9) encoding a codon-optimized ovine CLN5 transgene (oCLN5opt) driven by the chicken beta actin (CBh) promoter, produced by the University of North Carolina Gene Therapy Center Vector Core (NC, United States) as previously described (Mitchell et al., 2018). Vectors were formulated in 350 mM PBS containing 5% sorbitol, and titers determined by qualified droplet digital PCR.

Affected CLN5 ^−/− sheep (n = 3 per treatment age) were administered scAAV9/oCLN5 vector via concurrent ICV and IVT injection. For ICV delivery, stereotactic surgical procedures were used as described previously (Linterman et al., 2011; Mitchell et al., 2018). Bilateral ICV injections were performed in a volume of 400 µL per hemisphere (total volume 800 µL), delivered at a rate of 100 μL/min. Pre-symptomatic 3-month-old CLN5^−/− sheep received a moderate dose (MD; 2.9 × 10¹¹ viral genomes (vg)) via ICV delivery into the cerebral lateral ventricles, whilst early symptomatic sheep treated at 6 months or advanced symptomatic sheep treated at 9 months of age received a high-dose (HD; 3.3 × 10¹² vg).

After ICV delivery, anaesthetized sheep were repositioned for IVT administration as previously described (Murray et al., 2021). A total dose of 6.5 × 10¹⁰ vg scAAV9/oCLN5 was injected in a volume of 100 µL into the vitreous humor of the left eye. The right eye served as an internal control and was not treated. All ICV/IVT treated animals were sacrificed at a pre-determined timepoint (24 months of age) for post-mortem analysis.

General physical health and neurological clinical assessments were performed monthly on the treated CLN5^−/− sheep and age-matched cohorts of healthy CLN5^+/− and untreated CLN5^−/− sheep by two independent blinded investigators. Scores for each of the ten parameters in the ovine Batten disease rating scale (oBDRS) ranged from 4 to 0 (normal to abnormal), giving a total score of 40 (Mitchell et al., 2018). Cranial nerve function, mentation, gait, head carriage and postural traits, as well as manifest tremor or seizure onset, were reported. Live weight data and body condition scores were also collected.

As all treated CLN5^−/− sheep received IVT injections, the visual domain of the oBDRS was particularly exploited. Visual acuity was assessed through physical examination and in the field. Sheep were graded from 4 to 0 based on the following ten traits: head tilt, response to shadows, visual tracking, menace response, dazzle response, corneal/palpebral reflex, pupillary light reflex, funduscopic changes, electroretinography response, and hitting objects (Supplementary Table S2). In the event of a discrepancy between traits, sheep received the lowest possible grading. The only exception was when a sheep exhibited only a single reduced menace response, dazzle response, corneal reflex or pupillary light reflex (in one or both eyes) but no other visual deficits. In this situation, the sheep would score 3.

Visual and cognitive decline was assessed monthly in a previously described closed-field maze under daytime photopic light (Mitchell et al., 2018). Treated CLN5^−/− sheep and age-matched cohorts of healthy CLN5^+/− and untreated CLN5^−/− sheep individually negotiated the maze five times on each testing day to join conspecifics in the end pen. Sheep were allowed 2 min to complete the maze and the time taken to traverse was recorded. Testing continued monthly until sheep were unable to complete the maze.

Mixed response ERG recordings were obtained from each eye of treated sheep as previously described (Murray et al., 2021). Dark adapted b-wave amplitudes were compared with historical data from healthy CLN5^+/− (n = 6) and untreated CLN5^−/− sheep (n = 6) (Russell et al., 2021).

Computed tomography (CT) scans were performed on treated sheep every two to 4 months as previously described (Russell et al., 2018). Helical slices were obtained at 1 mm intervals, 120 kV, 100 mA, 2 s rotation time on a GE HD750 CT scanner (GE Healthcare, Hyugo, Japan). Three-dimensional (3D) modelling and intracranial volumetrics were performed using the 3D slicer 4.3.1 freeware (http://www.slicer.org; RRID:SCR_005619) (Federov et al., 2012; Russell et al., 2018).

Sagittal sheep brain sections were obtained as previously described (Oswald et al., 2005). In brief, sections were blocked, 30 min with 1% H₂O₂ in PBS (pH 7.4) containing 0.3% Triton X-100 (PBST) and then 1 h in 15% normal goat serum (NGS) in PBST prior to overnight incubation in primary antibody diluted in 10% NGS in PBST at 4°C. The following primary antibodies were used: rabbit anti-cow glial fibrillary acidic protein (GFAP, 1:5000; Agilent Cat# Z0334, RRID:AB_10013382) to detect activated astrocytes, a biotinylated form of the α-D-galactose specific isolectin I-B4 from Griffonia simplicifolia (GSB4, 1:500; Vector Laboratories Cat# B-1205, RRID:AB_2314661) for microglia and rabbit anti-sheep CLN5 (1:500; Viraquest Inc. Cat# R19122) for CLN5 protein expression. A biotinylated goat anti-rabbit IgG secondary antibody (1:1000, Sigma-Aldrich Cat# B7389, RRID:AB_258613) was applied for 4 h at room temperature, followed by ExtrAvidin peroxidase (1:1000, Sigma-Aldrich Cat# E2886, RRID:AB_2620165) for 4 h. Staining was visualized by incubation in 0.5 mg/mL 3, 3′-diaminobenzadine (DAB; Sigma-Aldrich Cat# D5637) and 0.01% H₂O₂ in PBS. Sections were mounted in a solution of 0.5% gelatine and 0.05% chromium potassium sulphate on glass slides, air-dried, dehydrated in 100% ethanol, cleared in xylene and coverslips mounted with DPX (BDH Chemicals, Poole, England).

Adjacent sections were processed through a standard cresyl violet Nissl stain to detect neuronal cytoarchitecture (Oswald et al., 2005). A parallel set of unstained sections was mounted, air-dried, and coverslipped with glycerol for fluorescent storage body analysis. Historical 50 µm brain and spinal cord sections from healthy control CLN5^+/− (n = 3) and diseased CLN5^−/− (n = 3) sheep were included in all staining runs as negative and positive controls.

Eye globes were enucleated at the time of death, separated into anterior and posterior segments, fixed in 10% formalin, 2 h, and sent to Gribbles Veterinary Pathology (Christchurch, New Zealand) for post-fixation in Bouin’s solution (Sigma-Aldrich Cat# HT10132), for 4 h followed by wax embedding. Retinal paraffin sections were cut at 3 μm, mounted and a subset stained with Hematoxylin and Eosin (H + E) histological stain by Gribbles Veterinary Pathology for analysis of retinal thickness. A parallel subset of unstained retinal sections was coverslipped in glycerol to assess fluorescent storage body accumulation.

Digital images of CLN5, GFAP, GSB4, Nissl-stained and unstained brain sections were obtained with a Nikon Digital Sight DSFi1 camera attached to a Nikon Eclipse 50i model microscope (Nikon Instruments Inc., Tokyo, Japan) utilizing NIS-Elements Software (Nikon Instruments; RRID:SCR_014329).

CLN5-positive somata and fibres were semi-quantified (from zero to 100 CLN5-positive somata/0.57 mm² field) in 20 fields for 11 distinct brain regions within each treated sheep hemisphere and 25 fields for three spinal cord regions (cervical, thoracic and lumbar). At least 25 thickness measurements were taken through three different cortical regions on Nissl-stained sections (Oswald et al., 2005). A set of 10 non-overlapping red-green-blue (RGB) images were captured at each sampling site per hemisphere for each immunostain, or with a 450–490 excitation/510 emission filter set for observation of fluorescent storage bodies.

Images were analyzed with ImageJ software (version 1.52p; National Institutes of Health (NIH), Bethesda, MD, United States; RRID:SCR_003070). Red bandwidth filters were applied, and a constant threshold value was used for each image set such that positively stained structures with low reactivity were still selected, but not background staining in regions of high reactivity. Results are presented as the percentage of immunoreactivity or fluorescence per sampled area.

Twenty total retinal thickness measurements per eye were taken from the surface of the nerve fiber layer (NFL) to the base of the retinal pigment epithelium (RPE) in both the central retina (within 5 mm of the optic nerve head) and the peripheral retina (10–20 mm from the optic nerve head). The number of photoreceptor nuclei were counted in 20 vertical columns through the outer nuclear layer of hematoxylin and eosin-stained retinal sections for each eye. In addition, ten images from the central retina were collected from unstained sections for each eye and thresholding analysis on ImageJ was used to determine the percentage of fluorescence per sampled area. The RPE and photoreceptor inner segment/outer segment layers were excluded from storage body analysis due to the endogenous fluorescence present in these cellular layers in the normal sheep eye.

All statistical analyses of neuropathological data were performed on GraphPad Prism software (v 9,0.0, GraphPad, La Jolla, CA, United States; RRID:SCR_002798). For most analyses, results were reported as mean ± the standard errors of the mean (SEM). Differences were considered as statistically significant where p < 0.05.

For the longitudinal ERG study, b-wave amplitudes were measured for each eye using the provided software (Eickemeyer). Animals were grouped by treatment and the repeated measurements were allocated into age-groups. ERGs were compared between treated and untreated eyes and over time using linear mixed regression models, with the lme4 package in R studio (v4.0.5; RRID:SCR_015654) (Bates et al., 2015; R Core Team, 2021). For each genotype, ERG was modelled independently as a function of age, treatment, and age-by-treatment interaction, with a varying intercept and slope for each sheep.

For the neuropathological studies, means (% area stained, cortical and retinal thickness, ONL count) and the corresponding SEM were computed for each brain region for each animal. These are presented in Supplementary Tables S3–S9. The means were used in a one-way Brown-Forsythe and Welch ANOVA followed by multiple unpaired t-tests assuming unequal variances and using a false discovery approach with the two-stage linear step-up procedure of Benjamini, Krieger and Tekutieli with a Q = 0.05 to test each region separately for differences between control, treated and untreated CLN5^−/− cohorts.

Bilateral ICV and unilateral IVT delivery was possible for all animals in the study. The treatment was well tolerated, and no safety concerns were identified with the delivery method or dose. In-life data were collected over the study before treated sheep were sacrificed for post-mortem analysis at a pre-determined timepoint of 24 months of age.

All the ICV/IVT treated animals showed steady weight gains over their first year, peaking at 16 months of age (Supplementary Figure S1). Two pre-symptomatic (1124/18, 1128/18) and two early symptomatic (1151/18, 1157/18) treated sheep reached healthy adult weights of 65–69 kg. However, the weights of advanced symptomatic treated sheep tracked closely with untreated CLN5^−/− controls, and one pre-symptomatic treated sheep (1102/18) was considerably lighter than the average CLN5^−/− sheep over her lifetime.

The ovine Batten disease rating scale (oBDRS) (Mitchell et al., 2018) was used to clinically assess treated sheep (Figure 1). Age-matched untreated CLN5^−/− historical and concurrent controls demonstrated the natural progressive disease course and were euthanized at 18.7 ± 1.0 months of age, with oBDRS scores of 10–15. Age-matched healthy control CLN5^+/− sheep maintained oBDRS scores of 40 for the duration of the study.

Prior to treatment, pre-symptomatic sheep were phenotypically normal. Following treatment, they had an initial small decline in average oBDRS score, attributed to a progressive reduction in menace response, indicative of vision loss, in the untreated eye (Figure 1B, green). Sheep 1124/18 remained highly alert at 24 months of age and the only clinical deficit was a reduced menace response, resulting in difficulty negotiating stairs. The other two sheep in this cohort (1102/18 and 1128/18) lost their menace response in both eyes by 21–22 months, startle response by 22 months, and were unaware of human approach to 5 m by 24 months. Although these sheep had mild signs of reduced mentation and capability (e.g., confusion, somnolence, and reduced herding) and struggled to negotiate stairs, they did not have any further signs of manifest clinical disease.

Prior to ICV/IVT injection of scAAV9/oCLN5, the early symptomatic sheep had a mild clinical phenotype, including a slight head tilt and/or low head carriage, reduced menace responses, wariness of shadows, and crouching when travelling through a confined gateway; all indicative of the onset of vision loss. Following treatment, these animals remained clinically stable until sacrifice (Figure 1B, orange) with an average oBDRS score of 38 at 24 months of age (18 months post treatment). Deviations from normal were attributed to a loss of vision in the untreated eye. Otherwise, these sheep were highly alert, responsive to stimuli (loud noises, approaching humans and food rewards), and highly interactive with their cohorts.

Advanced symptomatic sheep had an overt clinical phenotype prior to treatment, displaying low head carriage and difficulty negotiating stairs. Visual deficits were apparent, and their herding instinct was diminished. Following treatment, the average oBDRS score for this cohort remained stable, with a pretreatment score of 35 and a score of 34 at 24 months old (15 months post treatment) (Figure 1B, grey). These sheep showed no further advancement in disease symptoms from enrolment.

Analysis of the visual domain of the oBDRS showed that treatment with high-dose ICV/IVT scAAV9/oCLN5 sustained visual function in both the early and advanced symptomatic sheep compared with age-matched untreated CLN5^−/− sheep (Supplementary Figure S2). Efficacy was reduced for pre-symptomatic sheep that received an equivalent IVT dose but a lower ICV dose. Pre-symptomatic sheep 1102/18 and 1128/18 were classified as functionally blind (visual score ≤1) at 24 months of age and 1128/18 was actively running into objects (visual score 0).

Maze testing corroborated these findings (Supplementary Figure S3). After a period of habituation to learn the maze task, early symptomatic treated sheep demonstrated sustained cognitive and visual function, completing the maze in an average time of 28.8 ± 4.9 s over their lifetime, which was comparable to that of healthy CLN5^+/− control sheep (27.0 ± 3.1 s). Similarly, two out of the three advanced symptomatic treated sheep also completed the maze in an average of 27.3 ± 2.3 s over their lifetime. Times for the third animal in this cohort (1138/18) only slowed in the final two tests at 22 and 24 months of age, when this sheep failed to traverse one and two runs out of five respectively. In contrast, untreated CLN5^−/− sheep began to fail the maze from 19 months of age and could no longer participate from 21 months of age due to akinesia or confusion.

The pre-symptomatic sheep who had received an identical IVT dose but a ten-fold lower ICV dose than the other treatment cohorts demonstrated the lowest treatment effect. Only one pre-symptomatic treated sheep (1124/18) completed the maze with an average time of 28.3 ± 4.5 s over her lifetime. As indicated above, the other two sheep in this cohort lost vision in one or both eyes and failed to complete the maze fully from 22 to 24 months of age.

Longitudinal ERG (Russell et al., 2021) was also performed to monitor retinal physiology and function (Figure 2). IVT delivery of scAAV9/oCLN5 slowed stereotypical retinal dysfunction in both the early symptomatic and advanced symptomatic treated sheep. Their untreated eyes declined over time in line with data from untreated CLN5^−/− animals and, although there was a general trend towards loss of b-wave amplitude over time, the average rate of decline in the treated eye was almost half that of the untreated eye (Supplementary Table S10).

Despite receiving an identical IVT dose, but ten-fold lower ICV dose, retinal dysfunction was not halted in pre-symptomatic treated sheep. The average ERG amplitude from their treated eye initially tracked similarly to those of healthy control CLN5^+/− sheep, but over time declined steadily, and by 24 months of age it was similar to the untreated eye and close to or extinguished in all three sheep in this cohort.

Longitudinal computed tomography (CT) scanning (Russell et al., 2018) was performed on all treated sheep and compared with historical data from healthy CLN5^+/− and untreated CLN5^−/− sheep (Figure 3).

Pre-symptomatic treated animals had normalised intracranial volumes at baseline (5 months) with a steady average gain of 3.9 ± 1.3 mL over their lifetime (Figure 3B, green). Early symptomatic treated sheep had stable intracranial volumes from baseline to 24 months, with volume changes ranging from −0.6 to +1.6 mL (Figure 3B, orange). Similarly, advanced symptomatic treated sheep maintained relatively stable brain volumes over their lifetime (Figure 3B, grey). One animal (1131/18) in the advanced symptomatic cohort displayed a slow decline in intracranial volume with a total loss of 4.4 mL from baseline to 24 months of age, but the other two animals in that cohort (1138/18 and 1170/18) had mild gains of 0.7 mL and 1.1 mL respectively.

Overall, the volume increases, or stabilisation observed in the majority of the ICV/IVT-treated sheep contrasted the rapid atrophy seen in untreated CLN5^−/− sheep, which causes an average loss of 9.4 mL of intracranial volume between 3 and 19 months of age.

All ICV/IVT treated sheep were euthanized at 24 months of age for neuropathological analyses. Fresh brain weights of the ICV/IVT treated sheep were compared with average historical data collected from age-matched healthy control CLN5^+/− and untreated CLN5^−/− sheep (Table 1). Whilst treated brains were smaller than healthy CLN5^+/− controls mediolaterally and had some mild gyral flattening, there was no evidence of gross lesions (Supplementary Figure S4). They weighed between 73% and 89% that of mean healthy control CLN5^+/− brain weight and were 16–33 g heavier than the mean age-matched untreated CLN5^−/− sheep brain weight.

The biodistribution of CLN5 expression was analysed in the central nervous system of ICV/IVT treated animals by immunostaining with sheep CLN5 antibodies (Supplementary Figure S5). No CLN5 protein expression was seen in the untreated CLN5^−/− sheep brain tissue whereas endogenous CLN5 protein was widely expressed throughout the healthy control CLN5^+/− brain, particularly in the cortical neurons, pyramidal cells of the hippocampus and cerebellar Purkinje cells. Punctate vector-driven CLN5 expression was observed in the treated brains, throughout the cortex, cerebellum and hippocampus, and predominantly in morphologically neuronal cells. Strong transduction was also evident in the motor neurons in the ventral horn of the entire spinal cord length.

The number of CLN5-positive somata and fibres were semi-quantified for 11 distinct brain regions within the treated sheep brain hemispheres and three spinal cord levels (cervical, thoracic and lumbar) (Figure 4). Moderate to extensive CLN5 protein expression was observed in the motor, parieto-occipital and visual cortices and hippocampus of symptomatic sheep treated with scAAV9/oCLN5 at 6 or 9 months of age. Purkinje cells were the dominant population expressing CLN5 in the cerebellum whilst large subcortical structures, such as the thalamus, putamen or caudate nucleus had minimal expression. CLN5 expression was greater in the thoracic and lumbar regions than the cervical spinal cord.

A similar pattern of transduction was observed for the ICV/IVT sheep treated at a pre-symptomatic age (3 months) but with five to ten-fold less transduction efficiency (Figure 4). This correlated well with the fact that these animals received a ten-fold less ICV dose than the early and advanced symptomatic treated sheep.

Cortical thicknesses in the ICV/IVT-treated CLN5^−/− sheep were compared with age-matched healthy control CLN5^+/− and untreated CLN5^−/− sheep (representative images are shown in Figure 5A). Marked atrophy and neuronal loss was evident in all regions of the untreated CLN5^−/− cerebral cortex. In contrast, a distinct laminar distribution of cells was observed across the cortical mantle of all ICV/IVT scAAV9/oCLN5-treated brains. Treated sheep cortical thicknesses were reduced compared with those of the healthy control CLN5^+/− brain, ranging from 69% to 87% of normal thickness in the motor and parieto-occipital cortices and 60%–81% in the visual cortex (Figure 5B). However, all values were significantly greater than those of an age-matched untreated CLN5^−/− animal. The greatest treatment effect on cortical thickness was consistently seen in the early symptomatic animals treated at 6 months of age.

Despite the phenotypic differences observed between treated and untreated eyes, there was no significant difference in central retinal thickness when the treated eyes from the ICV/IVT sheep were compared with their untreated eyes (Figure 6). There was a trend towards a thicker retina in the treated eyes, with the largest difference between the two eyes seen in the advanced symptomatic cohort. Two sheep in this cohort (1138/18 and 1170/18) had a greater retinal thickness in their treated eyes compared with their untreated eyes (Figure 6B); while sheep 1131/18 had a thinner treated central retina, which corroborates with clinical loss of vision in this animal.

Results from the pre-symptomatic group were confounded by the lack of a central retina sample from the untreated right eye of sheep 1102/18, which was lost during processing for histology. This animal had a near healthy retinal thickness in the treated eye. There was no statistical difference in retinal thickness between eyes for the best (1124/18) and worst (1128/18) clinically performing sheep in this cohort.

In human and ovine NCL, retinal atrophy results from an almost complete loss of rod and cone photoreceptors (Weleber et al., 2004; Murray and Mitchell, 2022) hence photoreceptor nuclei were counted in the outer nuclear layer (ONL) of the sheep central retina. Counts showed that the number of nuclei was generally higher in both eyes of treated animals, with average counts of 4.6, 5.9 and 6.0 for the pre-, early and advanced symptomatic ICV/IVT treated animals respectively, compared with an average count of 2.4 ONL rows in the age-matched untreated CLN5^−/− eyes (Figure 6C).

The neuroinflammatory response in the ICV/IVT treated sheep was compared with age-matched healthy control CLN5^+/− and untreated CLN5^−/− sheep using an astrocytic marker (GFAP) and a microglial marker (GSB4) (Supplementary Figures S6, S7). Quantitative threshold analyses confirmed that ICV/IVT scAAV9/oCLN5 gene therapy suppressed neuroinflammation (Supplementary Figures S6, S7). Specifically, GFAP staining revealed a dense glial meshwork of hypertrophic activated astrocytes across the upper cortical layers of the untreated CLN5^−/− brain, which was not present in the scAAV9/oCLN5-treated brains (Supplementary Figure S6). Astroglial morphologies in the brains of treated sheep more closely resembled those seen in the control CLN5^+/− brain, being fibrous in the white matter and highly branched and protoplasmic with numerous processes in the cortical grey matter. Some hypertrophy and clusters of activated glia were detected in cortical layers II-III of treated sheep. However, the total GFAP immunoreactivity across the grey matter layers of the visual, parieto-occipital, and motor cortices of ICV/IVT treated animals was only half that seen in untreated CLN5^−/− animals of a similar age, indicating significant amelioration in disease-associated astrocytic activation.

A similar trend was seen with another neuroinflammatory marker, GSB4. A distinct reduction in activated microglia was observed in the cortical parenchyma of treated animals and this trend reached significance in the parieto-occipital and motor cortices (Supplementary Figure S7). There was no evidence of the clusters of GSB4-positive activated amoeboid microglia and hypertrophied brain macrophages that are seen in the untreated CLN5^−/− brain as two conspicuous bands within cortical layers II-III and V-VI (Supplementary Figure S7A).

Lysosomal storage was absent in healthy control CLN5^+/− sheep brain yet punctate, globular storage bodies were densely packed into the cortical neurons and glial-like cells of the untreated CLN5^−/− sheep brain, as well as in the perikarya of cerebellar Purkinje cells. The ICV/IVT treatment significantly reduced this storage body accumulation in the primary visual and motor cortices (Figure 7). This was particularly apparent in the pre-symptomatic and early symptomatic treated cohorts, whose cortices had half the lysosomal burden seen in age-matched untreated CLN5^−/− sheep.

Thresholding analysis was also performed on the treated and untreated retina from the ICV/IVT treated sheep (Supplementary Figure S8). In retina with fluorescent burden, distinct puncta were predominantly observed in the retinal ganglion cells; more diffuse storage accumulation was seen in the inner nuclear, outer nuclear, and plexiform layers. The retinal pigment epithelium (RPE) and inner segment/outer segment photoreceptor (IS/OS) layers were excluded from the analyses because high levels of endogenous fluorescence present in these layers in the healthy control CLN5^+/− sheep eye precluded a comparison.

Retinal fluorescence in both eyes of pre-symptomatic ICV/IVT treated sheep 1102/18 and 1128/18 was similar to that in untreated CLN5^−/− sheep (1.2%–2.2% vs. 1.30%, respectively: Supplementary Figure S8). In contrast, sheep 1124/18 had much less storage in the treated retina and more closely resembled a healthy control CLN5^+/− eye (0.22% vs. 0.16%, respectfully). Storage accumulation was lower in the retina of the treated eye compared with the untreated eye for all early symptomatic treated sheep although this did not reach statistical significance (Supplementary Figure S8B). Surprisingly, both eyes of the advanced symptomatic treated animals also had much lower levels of fluorescence compared with age-matched untreated CLN5^−/− sheep (Supplementary Figure S8B). While there was a trend for less storage body accumulation in the treated retina compared with the untreated retina for the advanced symptomatic sheep, statistical significance was not reached.