Enzyme replacement reduces neuropathology in MPS IIIA dogs

Enzyme replacement reduces neuropathology in MPS IIIA dogs

Neurobiology of Disease 43 (2011) 422–434 Contents lists available at ScienceDirect Neurobiology of Disease j o u r n a l h o m e p a g e : w w w. e...

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Neurobiology of Disease 43 (2011) 422–434

Contents lists available at ScienceDirect

Neurobiology of Disease j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y n b d i

Enzyme replacement reduces neuropathology in MPS IIIA dogs Allison C. Crawley a, Neil Marshall b, Helen Beard a, Sofia Hassiotis a, Vicki Walsh b, Barbara King a, Nicola Hucker b, Maria Fuller a, Robert D. Jolly b, John J. Hopwood a, Kim M. Hemsley a,⁎ a b

Lysosomal Diseases Research Unit, SA Pathology [Women's and Children's Hospital campus], North Adelaide, SA 5006, Australia Institute of Veterinary, Animal and Biomedical Sciences (IVABS), Massey University, Palmerston North 4442, New Zealand

a r t i c l e

i n f o

Article history: Received 27 December 2010 Revised 11 April 2011 Accepted 20 April 2011 Available online 29 April 2011 Keywords: Lysosomal storage disorder Sanfilippo syndrome Neuropathology Cerebrospinal fluid Recombinant enzyme Dog

a b s t r a c t There is no treatment for the progressive neurodegenerative lysosomal storage disorder mucopolysaccharidosis type IIIA (MPS IIIA), which occurs due to a deficiency of functional N-sulfoglucosamine sulfohydrolase (SGSH), with subsequent accumulation of partially-degraded heparan sulfate and secondarily-stored compounds including GM2 and GM3 gangliosides and unesterified cholesterol. The brain is a major site of pathology and affected children exhibit progressive cognitive decline and early death. In the present study, six MPS IIIA dogs received intravenous recombinant human SGSH (rhSGSH) from birth to either 8 or 12 weeks of age (1 mg/kg, up to 5 mg), with subsequent intra-cerebrospinal fluid injection of 3 or 15 mg rhSGSH (or vehicle) on a weekly or fortnightly basis to 23 weeks of age. All dogs completed the protocol without incident, and there was no clinically-relevant cellular or humoral immune response to rhSGSH delivery. Immunohistochemistry demonstrated rhSGSH delivery to widespread regions of the brain, and tandem mass spectrometry revealed an apparent dose-dependent decrease in the relative level of a heparan sulfatederived disaccharide, with near normalization of substrate in many brain regions at the higher dose. Secondarily-stored GM3 ganglioside and unesterified cholesterol, determined using histological methods, were also reduced in a dose-dependent manner, as was the number of activated microglia. We have demonstrated that pre-symptomatic treatment of this progressive neurodegenerative disorder via intracerebrospinal fluid injection of rhSGSH mediates highly significant reductions in neuropathology in this MPS IIIA model and clinical trials of this treatment approach in MPS IIIA patients are therefore indicated. Crown Copyright © 2011 Published by Elsevier Inc. All rights reserved.

Introduction The majority of lysosomal storage disorders (LSD) result from a mutation in a gene encoding a lysosomal enzyme, with subsequent accumulation of the substrate for the enzyme. There are more than forty LSD presently identified and two-thirds of these affect brain function. Clinically, LSD are highly debilitating, particularly when there is central nervous system (CNS) involvement, and for the majority of patients with CNS disease there is presently no treatment. Collectively, LSD affect Abbreviations: ABTS, 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid; CNS, central nervous system; CSF, cerebrospinal fluid; CV, co-efficient of variance; DAB, diaminobenzidine; ELISA, enzyme-linked immunosorbent assay; ERT, enzyme replacement therapy; GM3, ganglioside GM3; IC, intra-cisternal; IgG, immunoglobulin G; IgM, immunoglobulin M; IV, intravenous; LIMP-II, lysosomal integral membrane protein-II; LSD, lysosomal storage disorder; MPS, mucopolysaccharidosis; OCT, optimal cutting temperature; PBS, phosphate-buffered saline; RCA-1, ricinus communis agglutinin-1; rhSGSH, recombinant human sulfamidase; GlcNS-UA, glucosamine-N-sulfate uronic acid; SD, standard deviation; SGSH, sulfamidase; TNCC, total nuclear cell count; UV, ultraviolet. ⁎ Corresponding author at: Lysosomal Diseases Research Unit, 4th Floor Rogerson Building, SA Pathology, Women's and Children's Hospital campus, 72 King William Road, North Adelaide, SA 5006, Australia. Fax: + 61 8 8161 7100. E-mail address: [email protected] (K.M. Hemsley). Available online on ScienceDirect (www.sciencedirect.com).

one in 7700 live births (Meikle et al., 1999), although the advent of newborn screening for some disorders indicates that this is conservative (e.g. Spada et al., 2006; Duffner et al., 2009; Lin et al., 2009). Intravenous enzyme replacement therapy (ERT) is effectively used for treatment of non-neuronopathic mucopolysaccharidosis (MPS) type I, MPS II, Fabry and Pompe diseases and MPS VI (Kakkis et al., 2001; Schiffmann et al., 2001; Harmatz et al., 2005), however it is unlikely to be effective for treating CNS disease, as conventional doses of intravenously-delivered lysosomal enzymes do not readily gain access to the brain. Hematopoietic stem cell transplantation mediates cognitive improvements in some neurodegenerative LSD, but there is no published evidence demonstrating its clinical efficacy in patients with MPS III (Hoogerbrugge et al., 1995; Sivakumur and Wraith, 1999). One therapeutic option under active investigation is direct intracerebrospinal fluid (intra-CSF) ERT, with improvements in clinical signs and/or neuropathology reported in several different LSD animal models, e.g. MPS I dogs (Kakkis et al., 2004; Dickson et al., 2007, 2010), MPS IIIA mice (Hemsley et al., 2007, 2008, 2009a), Krabbe disease mice (Lee et al., 2007), late infantile neuronal ceroid lipofuscinosis mice (Chang et al., 2008), Niemann–Pick A mice (Dodge et al., 2009) and MPS VI cats (Auclair et al., 2010). We have previously undertaken intra-CSF enzyme delivery studies in adult MPS IIIA dogs with advanced disease (Hemsley et al., 2009b).

0969-9961/$ – see front matter. Crown Copyright © 2011 Published by Elsevier Inc. All rights reserved. doi:10.1016/j.nbd.2011.04.014

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Animal Welfare Act 1999. All procedures were also approved by the Children, Youth and Women's Health Service Animal Ethics Committee (South Australia) and conformed to the Prevention of Cruelty to Animals Act, 1985 and the NHMRC Australian Code of Practice for the Care and Use of Animals for Scientific Purposes, 2004. The dogs were bred, housed and maintained at a commercial research facility. Genotypes were established using previously described methods (Yogalingam et al., 2002). MPS IIIA animals were then randomly assigned to four groups, as described in Fig. 1. Tissues from four untreated normal or heterozygote dogs (designated “Unaffected” or “Normal”) aged 10 weeks, 15 weeks, 18 months and 68 months, and five untreated MPS IIIA dogs aged 0.5 weeks, 5.5 weeks, 10 weeks, 17 weeks, and 31 months were utilized as controls. Injections of rhSGSH/vehicle were administered IC with the animal in right lateral recumbency under general anesthesia. Following injection the animal was placed in sternal recumbency with the head held lower until recovery. Euthanasia and sample collection, including detailed brain dissection was performed 24 h after final IC rhSGSH/ vehicle dose, as described previously (Hemsley et al., 2009b). Briefly, at necropsy the brain was removed, weighed and the left hemisphere of the brain and other tissues, including portions of the spinal cord, were fixed in cold 4% paraformaldehyde (pH 7.2) or 10% buffered formalin. The right cerebral hemisphere was cut into approximately 10 × ~0.5 cm thick hemi-coronal slices prior to more detailed sample selection for biochemical investigations. The brainstem, spinal cord, cortex slice 4 (caudal; at the level of the caudal thalamus) and cortex slice 8 (rostral; at the level of the caudate/internal capsule) were dissected such that superficial (i.e. exposed to CSF) and deeper tissues were collected. Superficial tissue was collected to approximately 2 mm depth from the surface. Samples for electron microscopy were taken from selected sites and fixed for up to 48 h in 4% paraformaldehyde and 2% glutaraldehyde in phosphate buffer (pH 7.2) before transfer to phosphate buffer and routine processing into resin.

The MPS IIIA Huntaway dog exhibits a single base insertion, causing an immediate termination at 228 of 502 aa residues in N-sulfoglucosamine sulfohydrolase (SGSH; EC3.10.1.1; Yogalingam et al., 2002), thus no full length protein is transcribed. Disease-related pathology is severe in cerebral cortex, brainstem and cerebellum, with significant accumulation of secondarily-stored lipids and subsequent loss of Purkinje cells (Jolly et al., 2007). Macrophages containing vacuoles and membranous structures are also readily observed throughout the brain. The dogs exhibit progressive limb ataxia and hypermetria, observable by 3 years of age. In a proof-of-principle study, we established that weekly cisterna magna injection of recombinant human SGSH (rhSGSH) for up to 4 weeks in three adult MPS IIIA dogs results in delivery of enzyme to widespread areas of brain and spinal cord, with evidence of penetration of rhSGSH into deeper cortex and brain structures not directly in contact with CSF (Hemsley et al., 2009b). Relative levels of a heparan sulfate-derived disaccharide (GlcNS-UA), measured using tandem mass spectrometry, were lower compared to untreated MPS IIIA controls in many brain regions. At post-mortem it was evident that the dogs had mounted a significant cellular and humoral response to rhSGSH, with high titer antirhSGSH antibodies detected in both CSF and plasma (Hemsley et al., 2009b). In the present study, to minimize the immune response to rhSGSH in MPS IIIA dogs, we used a protocol in which we exposed newborn MPS IIIA puppies to weekly intravenous (IV) rhSGSH before initiating intra-cisternal (IC) ERT at 8–12 weeks of age (Fig. 1). A similar strategy was successfully employed in MPS VI kittens to enable longterm IV delivery of recombinant human 4-sulfatase into adulthood (Auclair et al., 2003). We have administered IC ERT to MPS IIIA dogs for 3 months, monitoring CSF and plasma for generation of antirhSGSH antibodies and an increase in cellularity, together with subsequent determination of the distribution and effect of rhSGSH on disease pathology by quantitative histology/immunohistochemistry and tandem mass spectrometry. Materials and methods

Enzyme production Animals RhSGSH was manufactured and purified by Shire Human Genetic Therapies and supplied at 9.9 mg/ml in 10 mM Na phosphate, 140 mM NaCl, pH 7.0. Dulbecco's phosphate buffered saline was used to dilute the enzyme to 1 mg/kg prior to IV injection. Elliotts B Solution (Ben Venue Laboratories Inc., Bedford, OH) was used to dilute

All breeding and experimental procedures were undertaken with the approval of the Animal Ethics Committee of Massey University (New Zealand) and conformed to The Code of Ethical Conduct for the Use of Animals for Teaching and Research as approved under the New Zealand

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Fig. 1. Treatment regimes in MPS IIIA Huntaway dogs. Some animals received weekly IV rhSGSH from birth (1 mg/kg up to 5 mg/dose; white arrows), prior to IC injection of 3 or 15 mg rhSGSH or buffer, commencing at 8–12 weeks of age. IC injections were administered weekly or fortnightly (black arrows) and euthanasia was 24 h after the final IC injection at 22–23 weeks of age. The number of animals in each group is shown in parentheses.

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rhSGSH to 3 mg/mL prior to IC delivery of 1 mL. The MPS IIIA IC buffer group received 1 mL Elliotts B Solution for IC delivery. Clinical monitoring and CSF analysis The general health of the dogs was monitored by daily observation. This was supplemented by full physical and neurological examination, and biochemical and hematological evaluation of blood samples before the study began, prior to each injection and prior to euthanasia. Blood (weeks 1–23) and CSF (weeks 12–23) samples were collected directly prior to next enzyme administration and were submitted to a commercial veterinary pathology laboratory for analysis. TNCC and red blood cell count in CSF samples were assessed manually using a counting chamber within 1 to 2 h of receipt of samples at room temperature, and protein levels were determined using standard techniques. White cell differentials on cytospins of 250 μL CSF were performed on 200 cells. Blood contamination in CSF was further evaluated using Multistix® urinalysis reagent strips (Siemens Healthcare Diagnostics Pty. Ltd., Bayswater, Victoria, Australia) which can accurately detect 10 rbc/μL. Detection of anti-rhSGSH antibodies in blood plasma and CSF Antibody titers were determined for heparinized plasma and CSF using a direct ELISA method (Hemsley et al., 2009b). Briefly, rhSGSHcoated multi-well plates (Immulon 4 HBX strips; Thermo Scientific, MA, USA) were blocked with TRIS–NaCl, 0.5% (w/v) gelatin and 0.2% (v/v) Tween-20; samples (100 μL) were then incubated for 2 h and detected with an horseradish peroxidise-conjugated rabbit anti-dog IgG (Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA) and ABTS substrate (Sigma, Australia). The resulting absorbance was read at 405–410 nm and titers expressed as the dilution for an absorbance greater than 2SD above the blank. Examination of neuropathological changes using light microscopy Standardized hemi-coronal slices of left cerebral hemisphere and a mid-line sagittal and parasagittal slice of cerebellum were either fixed in formalin and processed routinely into paraffin or were fixed in cold 4% paraformaldehyde (pH 7.2) in PBS for 24–48 h at 4 °C before being transferred to PBS and kept at 4 °C until cryoprotection and frozen sectioning. For cryoprotection, brain slices were placed in a solution of 30% sucrose in PBS overnight before freezing in OCT embedding medium, (Tissue Tek #4583, Sakura Finetek, Torrance, CA, USA) in a cryostat (Thermo Electron Corporation, Model Cryotome E, Runcorn, Cheshire, UK). Reagents for staining A monoclonal anti-LIMP-II antibody raised against a synthetic peptide (Mimotopes, Clayton, Victoria, Australia) mapping to the Cterminal region of human LIMP-II, was generously provided by Dr. E. Parkinson-Lawrence and Professor Doug Brooks (Lysosomal Diseases Research Unit, Adelaide, South Australia). Biotinylated lectin from Ricinus Communis Agglutinin 1 (RCA-1; Vector Laboratories, Burlingame, CA, USA), which recognizes β-D-galactose residues, was used as a histochemical marker for microglia. A mouse monoclonal (IgM) against GM3 (Clone GMR6) was purchased from Seikagaku Corporation, Tokyo, Japan. Affinity-purified polyclonal sheep anti-rhSGSH was generated inhouse (Meikle et al., 2006). Biotinylated donkey anti-sheep IgG (for rhSGSH; 1:1000) was purchased from Jackson ImmunoResearch (West Grove, PA, USA). Biotinylated donkey anti-mouse IgG (1:800, for LIMPII) and goat anti-mouse IgM (1:200, for GM3) were purchased from Chemicon International (Millipore, MA, USA) and Vector Laboratories, respectively. Filipin complex from Streptomyces filipinesis (F9765; Sigma, MO, USA) was used at a final concentration of 50 μg/mL.

Immunohistochemistry All procedures and post-staining image analyses were undertaken without knowledge of genotype/treatment status. LIMP II and rhSGSH immunostaining was carried out as described previously (Hemsley et al., 2009a, 2009b), with batch-staining performed. Briefly, sections were pre-treated with 0.5% hydrogen peroxide in methanol, underwent heatinduced epitope retrieval (Dako Cytomation target retrieval solution for LIMP-II; citrate-EDTA, pH 6.2, for rhSGSH) followed by incubation with the primary antibody (LIMP-II 1:800; rhSGSH 1:1000) and then a secondary species-specific biotinylated secondary antibody; the reaction was visualized using diaminobenzidine (DAB) enhanced with cobalt chloride (CoCl2). Histochemical staining for RCA-1 (adapted from Kiatipattanasakul et al., 1998) was performed by quenching endogenous peroxidase, as above, epitope retrieval with 0.05% trypsin (#T7409, Sigma) and incubation overnight in RCA-1 (5 mg/mL; 1:80). The reactions were visualized with DAB/CoCl2. Six-micron frozen sections underwent immunohistochemical detection of GM3 and histochemical visualization of unesterified cholesterol with filipin using the methods of McGlynn et al. (2004), using a mouse monoclonal to GM3 (#370695; Seikagaku), with visualization as above. Sections stained with filipin for visualization of unesterified cholesterol were stored at 4 °C in the dark until examination on an Olympus BX41 microscope with a fluorescent lamp and UV filter (435–485 nm). Image analysis Sections were viewed on an Olympus BX41 microscope and images collected using an Olympus Colourview III camera. For sections reacted with LIMP II, GM3 and RCA-lectin, a standardized region of cortex taken from a hemi-coronal section from each animal (approximately dorsolateral cortex for LIMP II and RCA-lectin, and dorso-medial cortex for GM3, both above the caudal hippocampus and caudal thalamus) was evaluated as follows: 8–15 fields of an average area of 0.14 mm 2 (200× magnification) from cortical ‘surface’ layers I-III, or ‘deep’ cortex (layers V-VI) were imaged, with all parameters/calibration constant for each comparative region and stain, within each stain batch. LIMP-II and GM3 images were analyzed using AnalySIS Lifescience Research (Olympus, Australia) software. Thresholding based on the optical density of positive immuno-staining was applied to the images in a consistent manner. Data are reported as mean% threshold area. For GM3 stained sections, freezing or ice crystal artifact, which appears as white non-staining holes, was calculated by thresholding and contributed to 4.8–6.3% (superficial cortex) and 4.5–9.6% (deep cortex) of section area. It was then removed from the total section area for final calculations of mean% area of staining. Manual counting of the number of activated microglia (per mm 2) was performed on RCA lectin-stained sections. Electron microscopy Samples of cortex and cerebellum were post-fixed in 1% osmium tetroxide, dehydrated and embedded in resin. One micron thick sections were stained with toluidine blue and examined by light microscopy and ultrathin sections from selected regions were cut and stained with 2% uranyl acetate/1% lead citrate. Sections were oriented so that the brain surface (cortex and cerebellum) or deep cortical white matter below Layer VI was evident. Morphological identification of cell types was carried out. Relative quantification of HS-derived oligosaccharides The relative amount of GlcNS-UA in tissue homogenates was determined via tandem mass spectrometry (Hemsley et al., 2009a).

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inclusions were detected in a 15 week old unaffected control. Finally, there was a marked increase in the number of activated microglia in MPS IIIA superficial cortex at 10 weeks of age, compared with agematched unaffected control tissue. The number of activated glia more than doubled in number by 23 weeks of age (Fig. 2C) and increased further by 31 months. Negligible numbers of activated microglia were found in 10 and 15 week old unaffected dogs, with slightly higher numbers counted in a 68 month old unaffected control.

Tissues were processed in seven batches with the same brain region from all the animals run in the same batch wherever possible. The intra-batch co-efficient of variation (CV) was determined by inclusion of eight replicates of MPS IIIA mouse brain, derivatized and analyzed at the same time as the test samples. The intra-batch CVs ranged from 3.7 to 11.3%; the inter-batch CV was 14%. Statistical analyses

Evaluation of the effect of IC rhSGSH therapy

Data from the high dose IV/IC animal was not included in any statistical analysis. CSF total protein, red blood cell counts and TNCC data were examined using a linear mixed model with compound symmetric covariance structure. Fixed effects of time, treatment and their interaction were tested. Red blood cell counts and TNCC data violated normality assumptions and were not amenable to transformation, so ranked data was used. Planned comparisons of each treatment at each time-point were performed and unadjusted pvalues are presented. Mass spectrometry data was analyzed using a one-way ANOVA with post-hoc Bonferroni correction. Histochemical staining data was analyzed using a linear mixed model with replicates clustered within an individual, and only the treatment effect was tested. Analyses were performed on log-transformed data for LIMP II and GM3 to satisfy normality assumptions, however, due to a large proportion of 0 values in the RCA-1 lectin data, a linear mixed model was run on the ranks of the data. In all cases, unadjusted p-values are presented and p b 0.05 was considered to be statistically significant.

Clinical observations All dogs were in good general health throughout the experimental protocol. Standard neurological tests were not carried out, given the difficulty in performing them on young pups, however all dogs appeared neurologically normal in appearance, gait and behavior and no neurological signs referable to MPS IIIA (primary cerebellar and cerebello-vestibular) were observed at any time. The procedures (IV, IC injections and associated anesthesia) were well-tolerated and weight gain was as anticipated for age/gender. CSF analysis A total of 81 CSF taps were performed, with two samples removed from analysis due to blood contamination. Total protein in CSF ranged from 0.06 to 0.22 g/L (normal reference range 0.1 to 0.25 g/L) over all four treatment groups. Total red blood cell count ranged from 0 to 348 cells/μL (normal reference range = 0). Elevations in red blood cell counts generally correlated with increases in total nucleated cell counts (TNCC), however elevated red cell counts were also observed without any other CSF changes outside normal reference ranges in all of the treatment groups (data not shown). A statistical evaluation of the effect of treatment on either protein concentration or red cell count with time (time treatment) between the low dose groups and controls was not significant (total protein, F16, 39 = 0.94; total red blood cells, F16, 39 = 0.66). Following low dose IV/IC rhSGSH, CSF samples from one animal (Ni) exhibited mild increases in TNCC prior to the 2nd, 3rd and 4th IC enzyme injections (13, 14, 15 weeks of age; 10–18 cells/μL), and a significant increase 24 h following the final IC enzyme delivery (170 cells/μL). The reference range for TNCC in normal canine cisternal CSF is b6 cells/μl (Chrisman, 1992). A moderate increase in TNCC was observed in the final sample from one dog following IC-only rhSGSH (67 cells/μL; Cha). This same dog also had a mild increase in cell count at 19 weeks of age prior to the 6th IC enzyme injection (8 cells/μL). One IC buffer-treated dog and one additional low dose IV/ IC rhSGSH-treated dog had levels of 12 and 7 cells/μL, respectively, 24 h following the final IC injection. The CSF TNCC for all remaining

Results Neuropathology in untreated MPS IIIA Huntaway dogs The relative level of GlcNS-UA was determined in four brain regions in MPS IIIA dogs aged 0.5 weeks to 31 months of age. At 0.5 weeks, GlcNS-UA in rostral and caudal cortex was nearly 500-fold elevated above levels seen in unaffected animals, with increasing amounts of this disaccharide observed in older dogs (Fig. 2A). In cerebellum and obex, grossly elevated GlcNS-UA levels were observed in all MPS IIIA animals evaluated, at all time-points. When storage pathology was examined histologically, the lysosomal membrane marker LIMP-II exhibited increased staining at 10 weeks of age, with nearly 7-fold normal levels recorded in the deep cerebral cortex. LIMP-II immunoreactivity increased with age to 23 weeks, and remained at a similar level at 31 months of age (Fig. 2B). Multiple electron-dense laminar bodies within membrane-bound inclusions were present in cerebellar Purkinje neurons and some cortical neurons in the 10 week old MPS IIIA animal (data not shown), with filipin staining revealing accumulating unesterified cholesterol in Purkinje and small molecular layer neurons (data not shown). No staining was seen in unaffected dogs, and no abnormal lipid-like

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Fig. 2. Age-related changes in disease markers in untreated MPS IIIA and normal dogs. A) Relative level of GlcNS-UA/mg protein in MPS IIIA and unaffected dog brain with age. B) LIMP II immunoreactivity in deep cerebral cortex (layers V–VI). C) Number of activated microglia in superficial cerebral cortex (layers I–III). In all graphs, each data point represents one animal.

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samples, including that from the high dose IV/IC animal, remained within the normal reference range. There was a significant time effect (F8,39 = 2.22, p = 0.047) but no significant time treatment interaction between the low dose groups and controls. However, the planned treatment comparisons at each time point did show that TNCC in IC rhSGSH-treated dog CSF was higher than in IC buffer (p = 0.02) or 3 mg IV/IC rhSGSH (p = 0.04) at 19 weeks of age. Antibody titers Plasma and CSF antibody titers are shown in Fig. 3. IC buffertreated MPS IIIA dogs exhibited negligible plasma titers (1:200– 1:3200). Two of the three 3 mg (low-dose) IV/IC-treated animals exhibited a similar range of titer values, with only minor elevations to 1:6400, generally during the first few weeks of IC treatment (Fig. 3A). The third animal from this group (Tw) exhibited increased plasma titers after the third IC injection (14 weeks of age), with a maximum titer of 1:25600 at 19 weeks of age (Fig. 3A). Both animals in the IC rhSGSH group exhibited plasma antibodies prior to the third IC injection (from 13 weeks of age), with the highest titer of 1:51200 (Fig. 3B). In the 15 mg (high dose) IV/IC dog, a significant elevation in titer (1:51200) was detected prior to the second IC injection (10 weeks of age), however subsequent samples were within the IC buffer-treated animal range, except for a minor elevation in the sample taken just prior to euthanasia (1:12800; Fig. 3C). With the exception of one dog, there was a complete absence of IgG antibodies in CSF at the lowest dilution evaluated (1:200; data not shown). Cha (IC rhSGSH) exhibited consistently elevated CSF titers after the third IC injection (Fig. 3D). A plasma sample taken following three IC injections of 3 mg rhSGSH into one adult MPS IIIA dog (Dog P4e) from Hemsley et al. (2009b) was used as a positive control sample and a titer of 1:2 × 10 6 was determined. A CSF sample from the same animal taken 24 h after the fourth IC injection of rhSGSH exhibited anti-rhSGSH antibodies with a titer of 1:1 × 10 6.

In one IC buffer-treated MPS IIIA dog, there was mild meningitis characterized by light infiltration of the leptomeninges with polymorph leucocytes and several microabscesses about the fourth ventricle. This is indicative of infection, most likely incurred at the last injection of Elliot's B solution, as CSF taken at that time had a cell count within normal limits. No other dog in this study exhibited any evidence of meningitis. No obvious reductions in Purkinje cell number were observed in buffer treated MPS IIIA dogs at 23 weeks of age. Immunolocalization of rhSGSH After scanning multiple brain regions, no positive immunoreactivity to rhSGSH was detected in sections from unaffected and IC buffer-treated MPS IIIA dogs (Supplementary Material, Figs. S1 and S2A,B,F,G; S3A,E). In contrast, 24 h following IC rhSGSH, positive staining was evident as dark punctate immunoreactivity within the cytoplasm of both neurons and glia, which were identified morphologically, in both low dose IV/ICand IC-rhSGSH-treated dogs (Supplementary Material, Figs. S1H,I), and in the high dose IV/IC animal (Supplementary Material, Figs. S1J). The extent of penetration of rhSGSH into the cortical, cerebellar and spinal cord parenchyma was determined by measuring the depth to which positively-stained puncta could be observed (Supplementary Table 1). Cerebellum and brainstem Intense, dose-dependent reactivity was evident in leptomeninges, with greater intensity seen in the tips of the cerebellar folia adjacent to the external cerebellar surface, extending only partway down fissures (Supplementary Material, Figs. S1C,D). In low dose IV/IC- and IC-rhSGSH groups, staining was found throughout the molecular and Purkinje cell layers, with faint staining present in limited numbers of granular cell layer neurons and intermittent Golgi cells and also in occasional glia in foliar white matter. In the high dose IV/IC animal,

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positive reactivity extended into the granule cell layer and was also present in significant numbers of glia scattered throughout foliar white matter. In many areas, negatively-staining Purkinje cells were surrounded by quite intense positive reactivity in the molecular layer. Positivity was only detected in cerebellar roof nuclei in the 15 mg rhSGSH dog. In brainstem, positive immunoreactivity was evident near the surface, extending deeper into the neural tissue, with positive puncta present in neurons and glia. Cerebral cortex Two regions of cortex were examined: caudally at the level of the caudal thalamus; and rostrally at the level of the optic chiasma. In caudal cortex from low dose IV/IC- and IC-only dogs, immunoreactivity was not uniformly distributed and was less intense overall than that seen in the cerebellum. Positive immunoreactivity was generally seen in macrophages in leptomeninges and in neurons, glia and perivascular macrophages in neuropil adjacent to the external brain surface or ventricular spaces, extending partway into the gray matter (layers I, II and sometimes superficially in layer III; Supplementary Material, Figs. S2C,D). Positive immunoreactivity extended into layer IV in one IC-only dog (Li; Supplementary Material, Fig. S2I). In all dogs, reduced reactivity was seen in the dorsal cortex region compared to the dorsomedial cortex. No obvious glial staining was present in white matter tracts, although quite intense reactivity was present in glia in corpus callosum ventral to the medial longitudinal fissure. Immunoreactivity was also noted superficially in the hippocampus and thalamus adjacent to the ventricles. Less extensive immunoreactivity was observed in rostral cortex than was observed caudally, with no neuronal staining observed in one IV/IC animal (Jl). Overall this was seen as fewer and less intense staining puncta, distributed even more superficially than in caudal

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cortex. Noticeably less overall immunoreactivity was observed in both cortex sections in one IC rhSGSH-only-treated dog (Cha). In the single high dose IV/IC dog, immunoreactivity in caudal and rostral cortex was more intense and more widely distributed. Positive puncta were present in neurons and glia extending down to cortical layer VI (Supplementary Material, Figs. S2E,J) and intermittent glia were stained in white matter. RhSGSH was detected in dentate neurons in hippocampus and thalamic neurons adjacent to the ventricular surface. Rare, very superficial staining was present in isolated regions of caudate adjacent to the ventricle. Spinal cord No immunoreactivity was observed in unaffected and buffertreated MPS IIIA dogs (Supplementary Material, Figs. S3A,B,E,F). In low dose IV/IC- and IC-only rhSGSH-treated dogs, positive immunoreactivity was present at the meningeal surface and extended partway into the white matter, with less extensive staining observed in lumbar cord compared to cervical regions. No immunoreactivity was present in ventral horn cells at either level (Supplementary Material, Figs. S3F, G), however intermittent glial staining was present in gray matter in some animals. Obvious reactivity was also present in the central spinal canal ependyma at both spinal cord levels in all low dose IV/IC-treated dogs (Supplementary Material, Fig. S3B), but not in either IC-only rhSGSH-treated dogs (Supplementary Material, Fig. S3C). Greater intensity and depth of penetration of rhSGSH staining was seen in the high dose IV/IC dog, with strong immunoreactivity at the meningeal surface extending into central gray matter. Fine immunopositive puncta were observed in the majority of ventral horn cells (Supplementary Material, Fig. S3H) in both cervical and lumbar cord, and in other small neurons, glia and perivascular macrophages

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Mean % LIMP II Immunoreactivity (y=log+1)

Deep

Superficial

A

Fig. 4. Effect of IC rhSGSH injections on LIMP II immunoreactivity. Photos in A–D show representative images from superficial cortex (layers I–III) in Normal, MPS IC buffer, MPS low IV/IC rhSGSH and MPS low IC rhSGSH dogs, with higher power insets. E–H show representative images from deep cortex (layers V–VI) adjacent to the white matter. Bar = 100 μm. Quantification of immunoreactivity in superficial (I) and deep (J) cortex, where each data point represents one animal. **** p b 0.0001, *** p b 0.001, compared to MPS IC Buffer; #### p b 0.0001, ## p b 0.01, # p b 0.05, compared to Normal.

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in the spinal cord gray (and white) matter. Strong immunoreactivity was also present in the central spinal canal ependyma (Supplementary Material, Fig. S3D). Trigeminal and cervical spinal cord dorsal root ganglion in the high dose IV/IC-treated dog exhibited positive puncta in multiple ganglion and surrounding satellite cells (data not shown). LIMP II immunoreactivity Low levels of LIMP II immunoreactivity were detectable in unaffected animals throughout the CNS, with faint puncta sometimes present in the cytoplasm of some cortical neurons (Figs. 4A, E). In contrast, in cortex and cerebellar regions in buffer-treated MPS dogs, moderate to intense dark punctate cytoplasmic LIMP II reactivity was present in most cell types (Figs. 4B,F) in both gray and white matter, although Purkinje cell immunoreactivity was absent. Reactivity was also absent in some cortical neurons. Quantitatively, there was a significant difference in the staining in both superficial and deep regions of the cortex, with much greater LIMP II immunoreactivity observed in buffertreated MPS IIIA dogs (Figs. 4I,J) compared with that seen in normal dog tissue. A significant treatment effect was observed in superficial cortex (F3,151 = 9.86, p b 0.0001) with immunostaining in both low dose IV/ICand IC-rhSGSH groups significantly lower than the buffer group. Low dose IC-rhSGSH did not significantly reduce LIMP II immunoreactivity in deep cortex, however staining in the 15 mg IV/IC dog was reduced compared with that seen in the 3 mg IV/IC group (Fig. 7C). GM3 immunoreactivity In normal dogs, faint puncta were seen at 1000× magnification in many neurons throughout the cortex (Figs. 5A,E), and more irregular

Normal

clumps of pale staining were present throughout white matter tracts. This was interpreted to indicate the presence of GM3 in unaffected oligodendroglia. Endothelial cell reactivity was also present in the pia and in blood vessels extending into the cortex. Similarly in unaffected cerebellum, faint punctate reactivity was present in small molecular layer neurons, and in glia at the junction of the molecular and Purkinje cell layers, however no positive staining was observed in Purkinje cell neurons. Conversely, in cortex from IC buffer-treated MPS IIIA dogs, variable but often intense, predominantly neuronal, cytoplasmic staining was evident throughout all cortical layers (I–VI), with increased intensity observed in large pyramidal neurons in layers III and V (Figs. 5B,F). In cerebellum, positive immunoreactivity was also clearly visible, particularly in distal dendritic processes from Purkinje cell neurons adjacent to the cerebellar surface and within cell bodies of Purkinje cells; however, the degree of reactivity within Purkinje cells ranged from negligible to intense punctate to globular cytoplasmic staining. Intense positive staining was present in golgi neurons in the granule cell layer; patchy staining was evident in other neurons and glia in the molecular layer, and staining was also seen in glia in white matter. Quantitatively, GM3 immunoreactivity was significantly higher in buffer-treated MPS IIIA dogs compared with normal dogs in both superficial and deep cortex regions (Figs. 5I,J). A significant treatment effect was observed in both regions (superficial cortex, F3,77 = 19.19, p b 0.0001; deep cortex, F3,77 = 10.35, p b 0.0001), with significant differences between the buffer group and all other groups in both regions. GM3 staining was reduced in the low dose IV/IC- and IC-rhSGSH groups to normal levels in superficial cortex, and to near normal levels in deep cortex (pN 0.05). Intermittent large pyramidal neurons with persistent strong immunoreactivity were sometimes detected in layer III in these dose groups. Similarly, in the cerebellum obvious reductions

MPS IC Buffer

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Mean % GM3 Immunoreactivity (y=log+1)

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A

Fig. 5. Effect of IC rhSGSH injections on GM3 immunoreactivity. Photos in A–D are representative images from superficial cortex (layers I–III) in normal, MPS IC buffer, MPS low IV/IC rhSGSH and MPS low IC rhSGSH dogs, with higher power insets. E–H show representative images from deep cortex (layers V–VI) adjacent to the white matter. Bar = 100 μm. Each data point represents one animal. **** p b 0.0001, *** p b 0.001, compared to MPS IC Buffer.

A.C. Crawley et al. / Neurobiology of Disease 43 (2011) 422–434

in immunostaining were present in the molecular layer, with rare persistent Purkinje cell dendritic staining and rare to mild immunoreactivity in Purkinje cell bodies. An almost complete absence of GM3 immunoreactivity was observed in both cortex and cerebellum samples from the high dose IV/IC dog (Fig. 7E). Quantitatively, GM3 immunoreactivity in deep cortex in the 15 mg IV/IC dog was less than that seen in the 3 mg IV/IC group and was indistinguishable from normal (Fig. 7F).

429

p b 0.0001). In the high dose IV/IC animal, RCA-1 staining in cortex and cerebellum appeared indistinguishable from the 15 week old untreated normal dog (Fig. 7H). When staining in deep cortex in the 3 mg and 15 mg IV/IC dogs were compared, fewer activated microglia were found following high dose treatment, where the number of activated microglia approached normal levels (Fig. 7I). Filipin staining of unesterified cholesterol

Lectin histochemistry Histochemical staining with RCA-1 lectin in both dorsal cortex and cerebellum of MPS IIIA buffer-treated dogs revealed strong positive staining in microglia, endothelial cells and perivascular macrophages in blood vessels throughout the neuropil, as described previously (Kiatipattanasakul et al., 1998). Microglia stained darkly and were variable in size with rounded cell bodies and minimal branching, characteristic of an activated state (Figs. 6B,F). Easily distinguishable from perivascular macrophages and endothelial cells, microglia were scattered throughout the gray and white matter, sometimes found as neuronal satellite cells. In untreated normal dogs, microglia were generally smaller, had minimal cytoplasm and were highly branched with long fine processes, consistent with resting microglia (Figs. 6A,E). In low dose IV/IC- and IC-rhSGSH dogs, the absence of large globular microglia was very noticeable in superficial cortex (layers II and III). In cerebellum, the overall intensity of staining was obviously reduced, but not to normal levels, with some larger and darker staining microglial cell bodies still present. These observations were confirmed by quantitative analysis of staining, which indicated a main effect of treatment in both superficial (F3,124 = 242.2, p b 0.0001) and deep cortex (F3,131 = 21.6,

Normal

MPS IC Buffer

No positive staining was detectable in unaffected dog tissue (Figs. 8A,F,K). In contrast, filipin reactivity was clearly evident in neuronal cytoplasm in buffer-treated MPS IIIA animals, with most intense staining seen in large layer V–VI neurons (Figs. 8B,L). Staining in superficial cortical layers was patchy and less prominent, with intermittent pyramidal-shaped neurons staining positively predominantly in layers II–III (Figs. 8B,G); reactivity was negligible in cortical white matter tracts. A striking reduction in filipin staining was observed in the cortical gray matter of low dose IV/IC-rhSGSH dogs (Figs. 8C,H,M), however, throughout most of the section, a band of persistent reactivity remained – approximately corresponding to layers III–VI – albeit with reduced intensity compared to staining in the MPS IIIA buffer-treated dogs (Figs. 8B,G,L). IC-rhSGSH dogs showed similar reductions in overall fluorescence in superficial cortical regions (Figs. 8D,I,N), with sparse positively-stained neurons in deep cortex adjacent to white matter. Even greater reductions in filipin reactivity were observed in the high dose animal, with no intensely staining neurons present (Figs. 8E,J,O). When cerebellar lobes VI to X were examined in unaffected dogs, only diffuse faint reactivity was observed, interpreted to be

MPS low IV/IC rhSGSH

MPS low IC rhSGSH

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500

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Mean # activated microglia /mm2

Deep

Superficial

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Fig. 6. Effect of IC rhSGSH injections on RCA-1 lectin staining of activated microglia. A–D show representative images from superficial cortex (layers I–III) in Normal, MPS IC buffer, MPS low IV/IC rhSGSH and MPS low IC rhSGSH dogs, with higher power insets. E–H are representative images from deep cortex (layers V–VI), adjacent to the white matter. Bar = 100 μm. Each data point represents one animal. **** p b 0.0001, *** p b 0.001, ** p b 0.01, compared to MPS IC Buffer; #### p b 0.0001, compared to Normal; ^^^^ p b 0.0001, compared to MPS IC rhSGSH.

A.C. Crawley et al. / Neurobiology of Disease 43 (2011) 422–434

I

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Mean % LIMP II Immunoreactivity (y=log+1)

A

MPS high IV/IC rhSGSH 15mg

Mean % GM3 Immunoreactivity (y=log+1)

MPS low IV/IC rhSGSH 3mg

Mean # activated microglia/mm2

430

Fig. 7. Effect of IC rhSGSH dose on disease markers. Photos depict LIMP II staining (A, B), GM3 immunoreactivity (D, E) and RCA-1 lectin staining (G, H) in the deeper layers (V–VI) of cerebral cortex of low (3 mg; A, D, G) and high (15 mg; B, E, H) dose dogs. Bar = 100 μm. Quantification of the staining of LIMP II (C), GM3 (F) and RCA-1 lectin (I) is shown. Each data point represents one animal.

autofluorescence, as it was different in appearance from positive reactivity observed in MPS IIIA dogs. In MPS IIIA buffer-treated dogs, while filipin staining was readily observable in Purkinje cell neurons, the pattern of staining was non-uniform, with intense staining in the majority of Purkinje cells in lobes XI and X, and less intense and sometimes absent staining in lobes VI to VIII. Negligible staining was observed in foliar white matter. In 3 mg IV/IC-rhSGSH-treated dogs, overall reduced staining intensity was observed in Purkinje cells with persistent, fainter staining of neurons in lobes IX and X. More extensive reductions were apparent in IC rhSGSH-treated dogs with rare positive cells seen in lobes IX and X. In the high dose IV/IC-rhSGSH animal, no positive staining was observed in any Purkinje cell neuron examined. Relative level of GlcNS-UA determined using tandem mass spectrometry The relative level of GlcNS-UA was negligible in 15 week old normal dog tissues (n = 4 samples; 0.03 ± 0.02 per mg total protein). In contrast, 23 week old buffer-treated MPS IIIA dog samples exhibited relative GlcNS-UA levels of 6.09 ± 0.20 (~ 200-fold normal; range 1.43–10.32; n = 94 samples, n = 3 dogs). Large, dose-dependent reductions in the relative amount of GlcNS-UA in rhSGSH-treated dogs were observed in surface regions of rostral and caudal cortex, cerebellum, brainstem and spinal cord (Fig. 9) with an average of 46(n = 47), 68- (n = 32) and 8- (n = 16) fold normal levels in the 3 mg IV/IC, 3 mg IC-only and 15 mg IV/IC-rhSGSH dose groups, respectively. Generally, a similar pattern was observed for ‘deep’ samples from these brain regions, however, the impact of rhSGSH on GlcNS-UA was

not as dramatic. In deep brainstem, smaller reductions in the disaccharide were observed in the 3 mg IV/IC group and no change was seen in the IC rhSGSH group when compared with the buffertreated MPS IIIA dogs; however, large reductions were seen in ‘deep’ brain samples in the 15 mg IV/IC dog to an average of 14-fold normal levels (n = 15). Relative GlcNS-UA levels were also determined in the medulla, proximal to the injection site and, again, the largest reductions were observed in surface regions (c.f. deep regions; data not shown). Dosedependent effects were noted. Reductions in GlcNS-UA were also recorded in hippocampus (to means of 29-, 58- and 1-fold normal in 3 mg IV/IC- or IC-rhSGSH and 15 mg IV/IC-rhSGSH groups, respectively; data not shown). While no or minimal reductions were observed in dorsal thalamus and caudate/internal capsule in the 3 mg IV/IC- and IC-rhSGSH groups, these same regions exhibited very large decreases in GlcNS-UA in the 15 mg IV/IC-treated dog (dorsal thalamus reduced to 38-fold normal and caudate/internal capsule reduced to 10-fold normal; data not shown). Of 33 brain regions analyzed, Cha from the IC-rhSGSH group had the highest relative GlcNS-UA levels in 20 brain regions compared with the other four animals in the low dose IV/IC- and IC-rhSGSH groups. Discussion The three-month IC-rhSGSH protocols used here initiated dosedependent reductions in both primary and secondary storage compounds and neuroinflammation (Table 1). Statistically significant

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MPS low IV/IC rhSGSH

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C

D

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I

J

K

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Fig. 8. Effect of IC rhSGSH injections on filipin staining of unesterified cholesterol in cerebral cortex. Photos in A–E show low power views of the dorsal cerebral cortex. Bar = 500 μm. Arrows in the left margin indicate approximate levels for higher power views of superficial (F–J; layers I–III) and deep cortex (K–O; layers V–VI). Bar = 50 μm.

reductions in neuropathology were observed in both surface and deep CNS tissues in MPS IIIA dogs receiving 3 mg IC-rhSGSH at euthanasia at 23 weeks of age. Further reductions in storage lesions were noted at the higher (15 mg) IC-rhSGSH dose. Immunolocalization of rhSGSH in various brain regions was consistent with the other analyses and demonstrated good distribution and penetration of rhSGSH, with maximal distribution seen in the 15 mg IV/IC-rhSGSH-treated dog. These observations support our original proof-of-principle findings in short-term IC rhSGSH-treated adult dogs (Hemsley et al., 2009b) where up to four IC rhSGSH injections resulted in widespread enzyme penetration into the brain and subsequent reductions in GlcNS-UA. However the present data provide a significant advance in our understanding of the impact of this treatment when undertaken longer-term in young dogs, and we have also elucidated the effect on a variety of secondary neuropathological changes seen in the MPS IIIA brain, namely secondary storage product accumulation and neuroinflammatory changes. Given that IC enzyme delivery began at 8–12 weeks of age, when significant neuropathological changes had already occurred in MPS IIIA dog brain, these outcomes are clinically-relevant. Studies of abortus material from human MPS III fetuses (Ceuterick et al., 1980) reveal the presence of residual bodies in utero. For the first time, we have shown that primary storage materials are also present in the MPS IIIA Huntaway dog brain within days of birth, with elevated GlcNS-UA recorded in all areas examined. Immunohistochemical studies performed on 10 week old MPS IIIA dog brain (earliest time evaluated using these methods) also revealed a significant increase in

LIMP-II expression and elevated numbers of activated microglia, with further increases in each marker with age. Therefore, given evidence of disease-related changes in developing MPS IIIA human and dog brain, the earliest application of treatments in humans will have the greatest likelihood of achieving positive clinical outcomes. We exposed newborn pups to repeated IV rhSGSH to prevent a severe immunological reaction following IC delivery of enzyme, as was observed in our proof-of-principle studies in adult MPS IIIA Huntaway dogs (Hemsley et al., 2009b); we were successfully able to extend the duration of IC treatment, with indications that this therapy could have continued without any detrimental effects. IC delivery of rhSGSH began much earlier in the course of disease in this study, with affected dogs receiving IC injections from 8 or 12 weeks of age (c.f. 21, 29 or 35 months of age in Hemsley et al. (2009b)). While the humoral immune system of the dog is believed to be mature at or around birth (reviewed in Felsburg, 2002), sequential development of proficiency in the response to different stimuli has been observed (Jacoby et al., 1969). Further, the magnitude of a humoral response to immunization differs between dogs treated as neonates or adults (Jacoby et al., 1969), therefore it is not yet clear whether IV pre-treatment is essential in order to avoid initiation of a robust immune response to subsequent IC delivery of rhSGSH in young MPS IIIA dogs. Total nucleated cell counts in CSF indicated that neither IV/IC- nor ICrhSGSH initiates significant infiltration of immune cells, a major observation in our previous studies in MPS IIIA dogs beginning treatment as adults. Further, while one of the dogs that was not pretreated with IV enzyme (Cha) exhibited significant elevations in anti-

A.C. Crawley et al. / Neurobiology of Disease 43 (2011) 422–434

GlcNS-UA (Peak area ratio/mg protein) Brainstem surface

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Fig. 9. Effect of rhSGSH treatment on the relative level of GlcNS-UA in MPS IIIA dog brain and spinal cord. Superficial and deep brain structures and spinal cord extending from rostral cortex to spinal cord from MPS IIIA dogs were examined for GlcNS-UA content. Rostral and caudal cortex samples included dorsal, lateral and ventral regions, cerebellum surface was collected from rostral, dorsal and caudal lobes, brainstem includes samples from superior colliculus and pons, spinal cord was collected at cervical, thoracic and lumbar levels. Each data point represents one sample. Samples from three young untreated MPS IIIA affected pups (0.5, 5.5, 10 weeks) were included for comparison. Negligible GlcNS-UA levels were detected in a 15 week old unaffected dog (mean GlcNS-UA = 0.03 ± 0.02, n = 4 samples; data not shown).

rhSGSH antibody titers in both plasma and CSF (the only dog to exhibit CSF antibodies in this study), the other IC rhSGSH dog exhibited only elevated plasma antibodies. Some of the data obtained are suggestive of a superior treatment outcome when puppies are pre-treated with IV rhSGSH. For example, the least extensive rhSGSH immunoreactivity was observed in rostral

and caudal cortex in Cha. Similarly, Cha exhibited little if any improvement in neuropathology when tissues were examined using EM (data not shown); filipin-staining in cerebral cortex was highest in this dog, and of 33 brain regions analyzed from the 5 animals in the 3 mg IV/IC- and IC-rhSGSH groups, Cha had the highest relative GlcNSUA levels in 20 brain regions compared with the other four animals.

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Table 1 Summary of effect of intra-cisternal rhSGSH on neuropathology in MPS IIIA dogs. MPS low IV/IC rhSGSH (n = 3)

MPS low IC rhSGSH (n = 2)

MPS high IV/IC rhSGSH (n = 1)

Age at treatment onset

12 weeks

12 weeks

8 weeks

Dose per brain weight†

34 μg rhSGSH/g brain

34 μg rhSGSH/g brain

172 μg rhSGSH/g brain

Surface — 2× N (68%A); deep — 5.6× N (32%A) Surface — b 1× N (90%A); deep — 3.7× N (72%A) Surface — 5.5× N (83%A); Deep — 55.3× N (58%A) AAANeuron layers I–III AA-AAANeuron layers IV–VI 85× N (66%A) Staining up to 1000 μm

Surface — 1.7× N (74%A); deep — 4.5× N (46%A) Surface — 1× N (86%A); deep — 2.3× N (83%A) Surface — 14.3× N (57%A); Deep — 62.6× N (53%A) AAANeuron layers I–III AA-AAANeuron layers IV–VI 85× N (62%A) Staining up to 1500 μm

Deep — 4× N (55%A) Deep–normal (100%A) Deep — 4.8× N (88%A) AAANear normalized in Layers I–VI 2xN (99%A) Staining throughout gyri (10,000 μm)

Cerebellum and brainstem surface GlcNS-UA 42× N (82%A) rhSGSH penetration Staining up to 600 μm

72× N (72%A) Staining up to 700 μm

19× N (92%A) Staining throughout folia (5770 μm)

Spinal cord surface GlcNS-UA rhSGSH penetration

24× N (74%A) Staining up to 1300 μm

4× N (97%A) Staining throughout cord (3500 μm)

Cortex LIMP II GM3 Activated microglia Unesterified cholesterol* GlcNS-UA# rhSGSH penetration

24× N (80%A) Staining up to 1500 μm

† calculated using 23 week-old MPS IIIA Huntaway dog brain weight of 87 ± 4 g (n=9). x N – fold normal. * Unesterified cholesterol: A mild reductions; AA moderate reductions; AAA extensive reductions. # Surface of caudal cortex.

These inferior treatment outcomes may have resulted from altered biodistribution of rhSGSH consequent to the formation of antibody complexes with rhSGSH. Therefore, while it is necessary to obtain additional data about the requirement for IV treatment of MPS IIIA dogs prior to starting intra-CSF rhSGSH delivery, it may be prudent, and indeed more clinically efficacious, to provide rhSGSH IV from birth which also enables non-CNS lesions to be treated. Our present findings support those obtained in MPS IIIA mouse studies, where dose-dependency has been described (e.g. Hemsley et al., 2007). Dose-dependent effects of intra-CSF delivery of lysosomal proteins have also been observed on survival in Krabbe mice (Lee et al., 2007) and in reductions in sphingomyelin accumulation in Niemann–Pick A mice (Dodge et al., 2009). Penetration of alpha-Liduronidase into the normal and MPS I dog brain has been reported to be dose-dependent (Kakkis et al., 2004; Dickson et al., 2007), however, at the doses used in these studies, there was no difference in the impact on glycosaminoglycan accumulation (Dickson et al., 2007). MPS I dog studies have demonstrated reductions in neuropathology following IC delivery of considerably lower doses of enzyme (Kakkis et al., 2004; Dickson et al., 2007). This may indicate that there are differences in the constitutive levels of the respective lysosomal enzymes that are required for normal neuronal function. Alternatively, it may indicate that the two enzymes exhibit significantly different half-lives. The estimated tissue half-life of rhSGSH following IV injection in MPS IIIA mice is likely to be b24 h based on data in Gliddon and Hopwood (2004), whereas for alpha-L-iduronidase, in vitro studies indicate a half-life of five days following uptake into human Hurler patient fibroblasts (Kakkis et al., 1994). Regardless of the reason(s) underlying this difference, we believe that based on the data generated in this study in Huntaway dogs, and in similar studies carried out in MPS IIIA mice, we are unlikely to be able to extend treatment intervals to quarterly, as has been proposed for MPS I patients (http://www.clinicaltrials.gov; NCT 00852358), based on data from MPS I dog studies (Dickson et al., 2007). The present study shows that 3 mg rhSGSH (equivalent to 34 μg rhSGSH/g wet brain weight; if 23 week-old dog brain weighs ~ 87 ± 4 g; n = 9) administered from an age at which lesions are mild but significant (12 weeks of age) was unable to ‘normalize’ either primary or secondarily-stored substrates (Table 1), indicating that the dose and weekly/fortnightly regimen used was insufficient to completely

remove substrates present at 12 weeks of age and subsequently maintain normal lysosomal function. Normalization of most aspects of disease pathology was achieved with very high dose (15 mg, equivalent to ~172 μg rhSGSH/g wet brain weight) enzyme treatment at fortnightly intervals (Table 1), however, treatment in this instance began at 8 weeks of age. Whether longer intervals between treatments will enable maintenance of a low level of lysosomal storage at this higher dose remain to be established. The ‘threshold’ below which primary storage needs to be maintained to enable and preserve normal clinical function is unknown at present. In previous studies in MPS IIIA mice we achieved ~80% reduction in GlcNS-UA levels across the brain/spinal cord, and observed improvements in neurological function (Hemsley et al., 2009a) at an equivalent dose of 200 μg rhSGSH/g wet brain weight (100 μg rhSGSH actual dose). We predict that at least this level of reduction in GlcNS-UA will therefore be required in MPS IIIA dogs and humans with this disease. Further, the level of primary storage that initiates subsequent pathological changes, e.g. accumulation of secondarily stored substrates and neuroinflammation in the case of MPS IIIA dogs and mice, is also yet to be established. Preliminary data collected in MPS IIIA mice suggests that alternative intra-CSF injection sites should be considered, with ventricular delivery potentially offering significant improvements in enzyme penetration and pathology amelioration when compared with cisternal injection (K Hemsley, unpublished data). Several studies in mouse models of other LSD have undertaken ventricular delivery of lysosomal enzyme, with impressive outcomes. For example, while Lee et al. (2007) performed single lateral ventricle injections in Krabbe mice and reported reduced neuropathology and increased lifespan, Chang et al. (2008) implanted osmotic pump devices, which enabled continuous but slow delivery of enzyme into the ventricular CSF in neuronal ceroid lipofuscinosis mice over the course of 2 weeks. This treatment strategy reduced tremor, reduced autofluorescent material and improved neuroinflammatory pathology. It remains to be determined whether bolus (via an indwelling subcutaneously accessed port) or continuous pump infusion provides the greatest benefit to the LSD brain, however, surgical methods enabling either strategy to be employed in patients are both available and feasible. In summary, n = 6 MPS IIIA dogs treated from a young age with ICrhSGSH exhibited dose-dependent improvements in all neuropathological parameters examined and near-normalization of storage

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pathology was seen in the one high-dose treated dog. The experimental protocol used here on a small group of dogs will enable us to undertake long-term clinical efficacy studies in MPS IIIA dogs to definitively determine the ability of this treatment strategy to delay or prevent the onset of functional changes and justify the application of this approach to patients. Supplementary materials related to this article can be found online at doi:10.1016/j.nbd.2011.04.014. Funding This work was supported by the Australian National Health and Medical Research Council [Grant #565074 to JJH, KMH and ACC) and Shire Human Genetic Therapies. Role of funding sources Shire Human Genetic Therapies provided the enzyme used in this study. The funding source did not have any role in study design, data collection, data analysis, interpretation of data, writing of the manuscript or in the decision to submit the paper for publication. Disclosure statement An international patent is held by JJH and others for mammalian sulfamidase and genetic sequences encoding it, for use in the investigation, diagnosis and treatment of subjects suspected of suffering from sulfamidase deficiency (US Patent No. #5,863,782). Acknowledgments We wish to thank Alastair Johnstone (Massey Uni.) for assistance with post-mortems, the Massey University IVABS clinic staff for the care they gave the dogs, Amanda Luck, Leanne Winner and Hanan Hannouche (LDRU) for tissue preparation, Nancy Briggs (Uni. Adelaide) for statistical advice and Dr Emma Parkinson-Lawrence and Prof Doug Brooks (UniSA) for providing the LIMP- II antibody. We acknowledge the generous provision of rhSGSH by Shire Human Genetic Therapies. References Auclair, D., Hopwood, J.J., Brooks, D.A., Lemontt, J.F., Crawley, A.C., 2003. Replacement therapy in Mucopolysaccharidosis type VI: advantages of early onset of therapy. Mol. Genet. Metab. 78, 163–174. Auclair, D., Finnie, J., White, J., Nielsen, T., Fuller, M., Kakkis, E., Cheng, A., O'Neill, C.A., Hopwood, J.J., 2010. Repeated intrathecal injections of recombinant human 4sulphatase remove dural storage in mature mucopolysaccharidosis VI cats primed with a short-course tolerisation regimen. Mol. Genet. Metab. 99, 132–141. Ceuterick, C., Martin, J.J., Libert, J., Farriaux, J.P., 1980. Sanfilippo A disease in the fetus— comparison with pre- and postnatal cases. Neuropadiatrie 11, 176–185. Chang, M., Cooper, J.D., Sleat, D.E., Cheng, S.H., Dodge, J.C., Passini, M.A., Lobel, P., Davidson, B.L., 2008. Intraventricular enzyme replacement improves disease phenotypes in a mouse model of late infantile neuronal ceroid lipofuscinosis. Mol. Ther. 16, 649–656. Chrisman, C.L., 1992. Cerebrospinal fluid analysis. Vet. Clin. North Am. Small Anim. Pract. 22, 781–810. Dickson, P., McEntee, M., Vogler, C., Le, S., Levy, B., Peinovich, M., Hanson, S., Passage, M., Kakkis, E., 2007. Intrathecal enzyme replacement therapy: successful treatment of brain disease via the cerebrospinal fluid. Mol. Genet. Metab. 91, 61–68. Dickson, P.I., Hanson, S., McEntee, M.F., Vite, C.H., Vogler, C.A., Mlikotic, A., Chen, A.H., Ponder, K.P., Haskins, M.E., Tippin, B.L., Le, S.Q., Passage, M.B., Guerra, C., Dierenfeld, A., Jens, J., Snella, E., Kan, S.H., Ellinwood, N.M., 2010. Early versus late treatment of spinal cord compression with long-term intrathecal enzyme replacement therapy in canine mucopolysaccharidosis type I. Mol. Genet. Metab. 101, 115–122. Dodge, J.C., Clarke, J., Treleaven, C.M., Taksir, T.V., Griffiths, D.A., Yang, W., Fidler, J.A., Passini, M.A., Karey, K.P., Schuchman, E.H., Cheng, S.H., Shihabuddin, L.S., 2009.

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