All study protocols were approved by an Institutional Animal Care and Use Committee.
Developmental time course in mice
Naglu−/− transgenic mice  (Naglu−/−, B6.129S6-Naglutm1Efn/J, The Jackson Laboratory, 00827, n = 5/age group) and WT controls (Naglu+/+, C57BL/6J, The Jackson Laboratory, 00664, n = 4–5/age group) were enrolled and euthanized at various ages spanning p3 to ≥ 12 weeks. Tissues were fixed in situ with 10% neutral buffered formalin for immunohistochemistry (IHC) analysis.
Systemic treatment in 12-week-old mice
Naglu−/− transgenic mice  (Naglu−/−, B6.129S6-Naglutm1Efn/J, The Jackson Laboratory, 00827) received either BMN 250 or vehicle twice weekly for 2 weeks via tail vein IV infusion. All mice, n = 4 male and n = 4 female per group (n = 16 mice total), were approximately 12 weeks of age at study start. Groups were randomized with respect to body weight and sex, although in previous studies no sex difference has been observed in the brain (data not shown). Mice were euthanized 1 day following the final infusion, and the brain was harvested into slices as indicated in Fig. 1. Brain slices 1 and 3 were processed for detection of NAGLU, HS and NRE, and β-hex. Brain slices 2 and 4 were fixed in 10% neutral buffered formalin processed for detection of NAGLU and LAMP2 via IHC.
NAGLU and LAMP2 IHC and image analysis in mice
NAGLU was assessed by staining with a NAGLU antibody shown previously to specifically detect rhNAGLU in Naglu−/− mouse brains after ICV administration . Lysosomal pathology was assessed by staining with a LAMP2 antibody also previously described .
Enzyme and activity assays in mouse studies
Recombinant human NAGLU-IGF2 fusion protein (BMN 250) was expressed in Chinese hamster ovary cells and purified, as described elsewhere . The purified protein (20 mg/mL) was stored frozen at − 80 °C in artificial CSF solution (Vehicle): 1 mM Na2HPO4/NaH2PO4, 148 mM NaCl, 3 mM KCl, 0.8 mM MgCl2, 1.4 mM CaCl2, pH 7.2. Prior to IV infusion, the enzyme was diluted, where necessary, to maintain the equivalent dosing volume throughout the studies. NAGLU enzyme activity in brain homogenate was determined using a synthetic fluorogenic substrate, 4-methylumbelliferyl (4MU)-N-acetyl-α-glucosaminide (EMD Millipore, Billerica, MA or Toronto Research Chemicals, North York, Canada), following a published protocol [5, 10] with minor modifications. β-hex activity was similarly determined using 4MU-N-acetyl-β-glucosaminide (EMD Millipore, Billerica, MA), as described previously. Activity is expressed as nanomoles of 4MU released per hour per total protein, which was estimated by Bradford assay with bovine serum albumin as standard.
Quantification of total and disease-specific heparan sulfate in mouse brain
Total HS and the disease-specific NRE of HS in homogenized brain samples were quantified using the Sensi-Pro assay, following previously published procedures [11, 12] and as previously described . Briefly, brain HS glycosaminoglycans were purified by anion exchange chromatography and digested with heparan lyases (IBEX Technologies). The depolymerization products were tagged and quantified by liquid chromatography-mass spectrometry. The measured quantity of total HS (internal disaccharides) and disease-specific NREs (trisaccharides, glucosamine-N-acetate alpha1,4 iduronate-2-sulfate alpha1,4 glucosamine 2-N-sulfate), referred to as A0I2S0  was expressed as picomoles per milligram of brain wet weight.
Measurement of vascular astrocytosis in mice
Formalin-fixed paraffin-embedded (FFPE) mouse brain sections were immunostained with goat anti-CD31 (AF3628, R&D Systems) alone or in co-stain with rabbit anti-GFAP (G9629, Sigma). Primary antibodies were detected using anti-goat immunoglobulin G (IgG) H&L (Alexa Fluor® 488) (A-11055, ThermoFisher Scientific) and anti-rabbit IgG H&L (Alexa Fluor® 555) (A-31573, ThermoFisher Scientific). For quantitative image analysis, whole mouse brain sections were scanned using a Leica Ariol slide scanning microscope with an HC PL APO 20×/0.7 objective and regions were extracted from every sample, one each from the cingulate cortex (cortical) and caudate putamen (subcortical). Using custom macros in ImageJ , the area of GFAP staining within a 3-μm distance from CD31-positive vessels was quantified to determine vascular astrocytosis. Statistics were analyzed in GraphPad Prism 6.
Cynomolgus monkey tissue samples
Frozen brain tissue from two healthy, untreated, male cynomolgus monkeys aged 3.9 years was acquired from Northern BioMedical Research (Norton Shores, MI). Cores were cut from superficial cerebral cortex, cerebral cortex, striatum, thalamus, midbrain, occipital cortex, medulla, and cerebellum and transferred to pre-chilled 4% paraformaldehyde in phosphate-buffered saline and fixed for 48 h at 4 °C. Fixed cores were processed and embedded in paraffin. Tissues were co-immunostained for rabbit anti-CI-MPR (ab124767, Abcam) with either goat anti-CD31 (AF3628, R&D Systems) or mouse anti-NeuN (MAB377, Millipore). Primary antibodies were detected using anti-rabbit IgG H&L (Alexa Fluor® 488) (A-21206, ThermoFisher Scientific), anti-goat IgG H&L (Alexa Fluor® 555) (A-21432, ThermoFisher Scientific), or anti-mouse IgG H&L (Alexa Fluor® 555) (A-31570, ThermoFisher Scientific). For quantitative image analysis, whole mouse brain sections were scanned using a Leica Ariol slide scanning microscope with an HC PL APO 20×/0.7 objective and two regions were extracted from every sample, one each from the cingulate cortex and somatosensory cortex. Using Perkin Elmer Volocity (6.3), the percent of CD31-positive vessels that were also positive for CI-MPR was quantified. Statistics were analyzed in GraphPad Prism 6. Representative confocal images were acquired using a Leica TCS SP8 microscope with an HC PL APO 40×/1.30 or 63×/1.4 oil objective and 1-AU pinhole diameter.
Human tissue samples
FFPE human cortical tissue from healthy donors was acquired from the NIH Neurobiobank at the University of Maryland, Baltimore, MD, for a range of ages: 2–3 months (n = 3), 1 year (n = 3), 2 years (n = 2), 3–7 years (n = 3), and 14–15 years (n = 3). FFPE human cortical tissue was immunostained with rabbit anti-CI-MPR (ab124767, Abcam) and detected using Impress anti-Rabbit HRP-conjugated secondary antibody (MP-7401, Vector Laboratories) followed by DAB substrate solution (SK-4100, Vector Laboratories). Images were acquired using a Leica DM5000 light microscope with 40× 0.85NA HC Plan Apo and 100× 1.4NA HCX Plan Apo objectives. DFC 550 top-mount camera and Leica LASX software were used.
IV and ICV treatment in cynomolgus monkey
Healthy male juvenile cynomolgus monkeys 11–13 months of age and weighing approximately 1.4–1.9 kg were put on study (n = 7). Animals receiving ICV treatment (n = 5) were surgically implanted with ICV catheters in the left lateral ventricle for dose administration, and all animals were surgically implanted with intrathecal catheters in the lumbar spine for CSF sample collection. ICV and IV administration routes were approved under separate protocols; the protocols for both studies received approval by the Institutional Animal Care and Use Committee, and both studies were conducted in accordance with the United States Public Health Service’s Policy on Humane Care and Use of Laboratory Animals.
Drug administration to NHP
Animals were administered a single dose of vehicle or BMN 250. For animals receiving ICV treatment, approximately 2.5 mL of CSF was withdrawn via cisterna magna spinal tap for isovolumetric administration to minimize potential intracranial pressure changes. Animals receiving ICV administration were administered a single dose of vehicle (n = 2) or 73 mg (n = 3), the maximum feasible for ICV administration based on infusion volumes and drug concentration, of BMN 250 with an infusion rate of 0.5 mL/min for ~ 5 min. Animals receiving IV administration (n = 2) were administered a single IV dose of 200 mg/kg for a total approximate dose of 350 mg, the maximum feasible, of BMN 250 at a dose volume of 10 mL/kg and a rate of 3 mL/min. The maximum feasible dose was selected for the IV route to maximize the chance of detecting drug exposure in the CSF and CNS tissue.
CSF drug concentration in NHP
For ICV administered animals, pharmacokinetic samples were obtained from CSF from the lumbar spine at concentrations putatively close to the maximum (at the end of infusion and 0.5 h post-dose). For IV animals, CSF and plasma samples were collected and tested immediately prior to infusion and at 0.25, 1, 3, 6, 12, 24, 36, and 48 h post-dose. Samples were analyzed for BMN 250 concentration by electrochemiluminescent assay (ECLA), utilizing a biotinylated murine anti-IGF2 monoclonal capture antibody and ruthenylated goat anti-NAGLU polyclonal detection antibody in a sandwich format to detect BMN 250. The standard curve was generated using a 4-parameter logistic regression model. BMN 250 concentration in each sample was determined by interpolation from the standard calibrator curve and adjustment for sample dilution. The quantitative range for CSF and plasma assays was 8.23–2000 ng/mL.
CNS tissue biodistribution
At 48 h following dosing, animals were euthanized and specific tissues of the CNS were harvested and perfused. The 48-h time point for euthanasia was selected as the putative tissue Cmax. For determination of NAGLU tissue concentration, specimens of superficial and deep tissue, relative to the ventricle, from seven brain regions (cerebellum, cerebral cortex, medulla oblongata, midbrain, occipital cortex, striatum, and thalamus) and from three spinal cord regions (cervical, lumbar, and thoracic) were weighed and homogenized using a Precellys 24-Dual (Bertin Instruments, Montigny-le-Bretonneux, France). Tissues were analyzed for NAGLU concentration by ECLA and normalized to total protein. The IGF2 tag of BMN 250 is cleaved in lysosomes upon cellular uptake, and this tag was utilized to remove intact extracellular BMN 250 from samples using anti-IGF2 coupled magnetic beads. Quantitation of NAGLU in cynomolgus monkey brain and spinal cord tissue homogenates was performed using a sandwich ECLA with a rabbit anti-NAGLU polyclonal capture antibody and a ruthenylated goat anti-NAGLU polyclonal detection antibody. The standard curve was analyzed using a 4-parameter logistic regression model and the concentration of NAGLU in each sample was determined by interpolation from the standard calibrator curve and adjusted for sample dilution. The quantitative range was 4.92–200.0 ng/mL.
Because the doses administered ICV and IV were not the same, analyses are presented as dose-normalized: bioanalytical assay results were divided by total administered dose, such that comparisons were made on a per unit basis. Previous investigations (data not shown) with ICV and IV-administered BMN 250 to cynomolgus monkeys have shown dose proportionality following single and multiple doses in tissue, CSF, and plasma across the dose range studied here. Dose proportionality suggests dose-normalization is appropriate.
Cynomolgus monkey IHC and image analysis
Cores (54 mm) were cut from frozen brain slices of selected brain regions with the highest NAGLU concentration following IV administration (superficial medulla oblongata and superficial midbrain). These regions were selected to maximize the likelihood of detecting NAGLU across cell types. Cores were transferred to pre-chilled 4% paraformaldehyde in phosphate-buffered saline and fixed for 48 h at 40 °C. Fixed cores were processed for FFPE using the Leica TP1020 and sectioned into 7 μm sections onto charged slides. Sections were deparaffinized and rehydrated to water, incubated in 10 μg/mL proteinase K in Tris-EDTA at 37 °C for 20 min and on benchtop for 10 min, and blocked in 2% normal donkey serum, 0.1% bovine serum albumin, 0.3% triton in tris-buffered saline for 1 h at room temperature. Sections were co-stained using a rabbit polyclonal antibody against BMN 250 (made in house, 1:400) and CD31 (ab9498, 1:100) and incubated overnight at 40 °C. Primary antibodies were detected with anti-rabbit IgG (H + L) Alexa Fluor® 488 (A21206, ThermoFisher Scientific) and anti-mouse IgG (H + L) Alexa Fluor® 555 (A31570, ThermoFisher Scientific). Sections were mounted in Fluoromount-G with DAPI (17984-24). Images were acquired on a Leica SP8 confocal microscope and adjusted in Adobe Photoshop using identical parameters for all samples.