supplementary materials for · 2020-05-22 · volumes (cv) of pbs with 500mm nacl at ph 7.4. bound...
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stm.sciencemag.org/cgi/content/full/12/545/eaay1163/DC1
Supplementary Materials for
Brain delivery and activity of a lysosomal enzyme using a blood-brain barrier
transport vehicle in mice
Julie C. Ullman, Annie Arguello, Jennifer A. Getz, Akhil Bhalla, Cathal S. Mahon, Junhua Wang, Tina Giese, Catherine Bedard, Do Jin Kim, Jessica R. Blumenfeld, Nicholas Liang, Ritesh Ravi, Alicia A. Nugent,
Sonnet S. Davis, Connie Ha, Joseph Duque, Hai L. Tran, Robert C. Wells, Steve Lianoglou, Vinay M. Daryani, Wanda Kwan, Hilda Solanoy, Hoang Nguyen, Timothy Earr, Jason C. Dugas, Michael D. Tuck, Jennifer L. Harvey,
Michelle L. Reyzer, Richard M. Caprioli, Sejal Hall, Suresh Poda, Pascal E. Sanchez, Mark S. Dennis, Kannan Gunasekaran, Ankita Srivastava, Thomas Sandmann,
Kirk R. Henne, Robert G. Thorne, Gilbert Di Paolo, Giuseppe Astarita, Dolores Diaz, Adam P. Silverman, Ryan J. Watts,
Zachary K. Sweeney, Mihalis S. Kariolis*, Anastasia G. Henry*
*Corresponding author. Email: [email protected] (M.S.K.); [email protected] (A.G.H.)
Published 27 May 2020, Sci. Transl. Med. 12, eaay1163 (2020)
DOI: 10.1126/scitranslmed.aay1163
The PDF file includes:
Materials and Methods Fig. S1. Validation of LC-MS/MS methods and cellular experimental models. Fig. S2. PK of ETV:IDS in wild-type mice. Fig. S3. GAG reduction in fluids and peripheral tissue biodistribution of ETV:IDS in IDS KO mice. Fig. S4. PK of ETV:IDS in TfRmu/hu mice. Fig. S5. IDS deletion does not affect TfR levels in the brain. Fig. S6. Individual disaccharide analysis from the brain and CSF of dosed IDS KO; TfRmu/hu KI mice. Fig. S7. Characterization of FACS-isolated CNS cell types. Fig. S8. Analysis of ganglioside, BMP, and glucosylceramide levels in FACS-isolated CNS cell types after treatment with ETV:IDS. Fig. S9. Relationship between dose level and brain concentration of ETV:IDS. Fig. S10. Analysis of lysosomal lipids in CSF after chronic treatment with ETV:IDS. Table S1. LC-MS acquisition parameters information for the BMP and gangliosides assay. Table S2. LC-MS acquisition parameters information for GlcCer and GalCer assay. Table S3. LC-MS acquisition parameters information for eicosanoid assay. Table S4. LC-MS acquisition parameters for lipidomics assay in negative ionization mode. Table S5. LC-MS acquisition parameters for lipidomics assay in positive ionization mode. Table S6. Targeted lipidomic analysis of IDS KO; TfRmu/hu KI mice after repeated administration of ETV:IDS or idursulfase.
Legend for data file S1 References (75–88)
Other Supplementary Material for this manuscript includes the following: (available at stm.sciencemag.org/cgi/content/full/12/545/eaay1163/DC1)
Data file S1 (Microsoft Excel format). Raw data.
Materials and Methods
Expression and purification of recombinant human ETV:IDS and Fc:IDS
ETV:IDS were expressed as knob-in-hole bispecific proteins. ExpiCHO cells (Thermo Fisher
Scientific) were transfected with plasmid DNA encoding the TV Fc polypeptide and IDS fused
to an Fc polypeptide in a 1:1 plasmid ratio by weight using Expifectamine CHO transfection kit
according to manufacturer’s instructions (Thermo Fisher Scientific). Expression of recombinant
IDS:Fc was carried out as described above with the exception of wild-type Fc being used in
place of the TV.
ETV:IDS and Fc:IDS were subsequently purified from conditioned media by protein A affinity
chromatography (GE MabSelect SuRe). Once loaded, the column was washed with 10 column
volumes (CV) of PBS with 500mM NaCl at pH 7.4. Bound proteins were eluted with 50 mM
sodium citrate, 150 mM NaCl at pH 3.0 and immediately neutralized using 1 M arginine with
670 mM succinate buffer at pH 5.0. For ETV:IDS, protein A eluates were dialyzed in 20 mM
NaPO4 and 20 mM NaCl at pH 7.0 overnight and further purified by anion-exchange
chromatography over a HiTrap Q HP column (GE Healthcare). Briefly, after binding, the column
was washed with 10 CV of 20 mM NaPO4 and 20 mM NaCl at pH 7.0. Bound proteins were
eluted using a linear gradient of 20 mM NaCl to 500 mM NaCl, with 20 mM NaPO4 at pH 7.0.
For Fc:IDS, the protein A pool was further purified over Superdex200 (GE Healthcare) with 20
mM NaPO4 and 150 mM NaCl at pH 7.0. Homogeneity of ETV:IDS and IDS:Fc were assessed
by SDS-PAGE and analytical SEC-HPLC.
KD determination using Surface Plasmon Resonance
Affinities of ETV:IDS variants for hTfRapical were determined by surface plasmon resonance
using a Biacore T200 instrument. Streptavidin was immobilized on a Biacore Series S CM5
sensor chip and biotinylated hTfRapical (0.5 mg/mL) was captured for 15 seconds at a flow rate
of 10 ml/minute. ETV:IDS was buffer exchanged into HBS and a 3-fold serial dilution was
injected across the chip at a flow rate of 30 ml/minute. Binding response was corrected by
subtracting the response units (RU) from a flow cell capturing a control IgG at similar density.
TfR binding affinities were obtained either by 1:1 Langmuir model of simultaneous fitting of k
on and k off or by fitting the response at equilibrium against the ETV:IDS concentration using
Biacore™ T200 Evaluation Software v3.1.
In vitro IDS activity assay
The specific activity of idursulfase (produced by Shire and purchased through WEP Clinical and
Myoderm) and ETV:IDS variants was measured with a two-step fluorometric enzymatic assay
using an artificial substrate(75). Briefly, 20 µL of 1 mM 4-Methylumbelliferyl a-L-
idopyranosiduronic acid 2-sulphate disodium salt substrate (Carbosynth) diluted in assay buffer
(100 mM sodium acetate, 0.05% Triton X-100, pH 5.0) was mixed with 10 µL of 0.1 nM of
idursulfase or ETV:IDS. The first reaction was incubated for 4 hours at 37°C and terminated
with 0.2 M phosphate-citrate buffer, pH 5.0. The second reaction was carried out in the presence
of cell lysate from HEK293T IDS KO cells (described below) transiently transfected with human
a-iduronidase (IDUA), incubated for 16 hours at 37°C, and stopped with the addition of 0.5 M
sodium carbonate buffer, pH 10.5. Fluorescence of the reaction solution was measured
(excitation at 365 nm and emission at 450 nm). A 4-Methylumbelliferone standard curve was fit
by linear regression to calculate the amount of product and verified as less than 10% of total
substrate cleavage. Specific activity (nmol product/minute/nmol IDS) was calculated by dividing
the amount of product by the reaction time and molar amount of IDS.
IDS:Fc and IDS:IDS ELISAs and PK Analysis
ETV:IDS and idursulfase concentrations in mouse serum, liver, or brain lysate were quantified
using two sandwich ELISA formats. ETV:IDS was captured using an anti-Fc antibody (Abcam
ab124055 or Jackson Immunoresearch cat. no. 709-006-098) for the IDS:Fc ELISA format, and
IDS was captured using an anti-IDS polyclonal antibody (R&D Systems AF2449) for the
IDS:IDS format. Following incubation with diluted plasma, serum, or lysates, both ELISA
formats used a biotinylated polyclonal anti-IDS antibody (R&D Systems BAF2449) followed by
streptavidin-HRP for detection. Individual standard curves for ETV:IDS or IDS were fit using a
five-parameter logistic regression. Systemic clearance was determined using non-compartmental
analysis in either Phoenix WinNonlin (Certara, Inc.) or the Dotmatics Suite (Dotmatics
Knowledge Solutions). Half-lives were estimated from the terminal phase of plasma or serum
concentration profiles.
Liquid chromatography-mass spectrometry analysis of fGly
ETV:IDS or idursulfase were buffer exchanged into 50 mM ammonium acetate and final protein
concentrations were measured using a BCA assay (Pierce). Proteins were subsequently added to
Rapid Digest Buffer (Promega) with 5 mM TCEP and reduced for 45 minutes at 37°C.
Alkylation was performed with the addition of 12.5 mM iodacetamide for 60 minutes in the dark.
Chymotrypsin (0.5 µg, Promega) was added and left to digest at room temperature for 2 hours.
Digested samples were dehydrated via speed vacuum at 45°C for 1.5 hours. The dried pellet was
resuspended in 50 µL of milli-Q water with 5% acetonitrile and 0.1% formic acid and vortexed
for 10 minutes prior to LC-MS/MS analysis.
Six unique target peptides were generated by chymotrypsin digestion of IDS: endogenous
unmodified cysteine (Peptide 1, AQQAVCAPSRVSF, [M+2H]2+ m/z 682.3435, RT 4.4),
carbamidomethylation modification on cysteine (Cys-CAM) during in vitro IAA alkylation
(Peptide 2, AQQAVC(CAM)APSRVSF, [M+2H]2+ m/z 710.8537, RT 4.0), formylglycine (fGly)
(Peptide 3, AQQAVFGlyAPSRVSF, [M+2H]2+ m/z 673.3471, RT 3.9) and oxidized
formylglycine (oxo fGly) (Peptide 4, AQQAVFGly(O)APSRVSF, [M+2H]2+ m/z 681.3446, RT
4.0). A heavily labeled peptide (Peptide 5, AQQAVCAPSR(13C5, 15N)VSF, [M+2H]2+ m/z
685.3489, RT 4.4 ) (Tufts University Medical School, Boston, MA) as well as the alkylated Cys-
CAM version (Peptide 6, AQQAVC(CAM)APSR(13C5, 15N)VSF, [M+2H]2+ m/z 713.8598,
4.0) were used for peptide confirmation and semi-quantitation. All peptides’ m/z are 2+ charge.
fGly analysis was performed by liquid chromatography (Ultimate 3000, Thermo Fisher
Scientific) coupled to electrospray mass spectrometry (Q Exactive, Thermo Fisher Scientific).
For analysis, samples were injected on a Acquity BEH C18 1.7 mm 2.1x100 mm column
(Waters Corporation,) at a flow rate of 0.4 mL/minute with a column temperature of 40°C.
Mobile phases A and B were water with 0.1% formic acid, and acetonitrile with 0.1% formic
acid, respectively. A gradient was programmed as follows: 0.0–0.5 minutes hold at 5%B, 0.5–3.0
minutes from 5%B to 20%B, 3.0-6.0 minutes hold at 20%B; 6.0-6.5 minutes from 20%B to
50%B, 6.5-7.5 minutes hold at 50%B, 7.5-8.0 minutes 50%B to 80%B, 8.0-9.0 minutes hold at
80%B, 9.1 minutes back to 5%B and hold to 10 minutes. The mass spectrometer was used in full
mass scan from m/z 650 to m/z 750 at 140,000 resolution, AGC target at 1e6, max injection time
at 100 ms. Parallel reaction monitoring was used for peptide identification. All peptides were
detected with high mass accuracy for the parent mass within 5 ppm and confirmed by fragment
ions. Quantification was performed using TraceFinder (Thermo Fisher Scientific). The
percentage of fGly was calculated by dividing the peak area of peptide 3 by the sum of all
peptide area peaks (peptides 1-6) and multiplying the ratio by 100.
Generation of HEK 293T IDS KO cells
HEK 293T cells (ATCC) were transfected with CRISPR/CAS9 pCas-Guide-EF1a-GFP vector
(Origene) containing guide sequences targeting the second half of exon 1 in human IDS. Single
cell clones were analyzed for the presence of indels within the genomic sequence of IDS
following Guide-it Mutation Detection Kit (Clontech) per manufacturer instructions. Indel
positive cell lysates were subjected to an in vitro IDS enzyme assay using the IDS fluorogenic
substrate 4-methylumbelliferyl-alpha-iduronate (Carbosynth). Briefly, the in vitro activity assay
was performed as described above using cell lysate diluted in assay buffer (100 mM sodium
acetate, 10 mM lead acetate, 0.02% NaAzide, pH 5.0) as input. IDS activity in HEK293T
CRISPR clones was compared to idursulfase used as an assay standard, HEK293T wild type
(WT) lysates, and HEK293T cell lysates over-expressing IDS. Clones with enzyme activity
comparable to background signal were sequence verified after mini-Topo (Thermo Fisher
Scientific) cloning and confirmed as KO clones. Subsequent cell assays used three unique and
verified IDS KO clones and three independent batches of WT HEK293T cells.
Mass spectrometry analysis of GAGs
Quantification of GAGs in cells, fluids, and tissues was performed by liquid chromatography
(Shimadzu Nexera X2 system, Shimadzu Scientific Instrument) coupled to electrospray mass
spectrometry (Sciex 6500+ QTRAP, Sciex). For each analysis, sample was injected on a
ACQUITY UPLC BEH Amide 1.7 mm, 2.1×150 mm column (Waters Corporation) using a flow
rate of 0.4 mL/minute with a column temperature of 50°C. Mobile phases A and B consisted of
water with 10 mM ammonium formate and 0.1% formic acid, and acetonitrile with 0.1% formic
acid, respectively. A gradient was programmed as follows: 0.0–1.0 minutes at 85%B, 1.0–5.0
minutes from 85%B to 50%B, 5.0–6.0 minutes 50%B to 85%B, 6-8.0 minutes hold at 85%B.
Electrospray ionization was performed in the negative-ion mode applying the following settings:
curtain gas at 30; collision gas was set at medium; ion spray voltage at -4500; temperature at
450°C; ion source Gas 1 at 50; ion source Gas 2 at 60. Data acquisition was performed using
Analyst 1.6.3 (Sciex) in multiple reaction monitoring mode (MRM), with dwell time 30 msec for
each species. Collision energy at -30; declustering potential at -80; entrance potential at -10;
collision cell exit potential at -10. GAGs were detected as [M-H]- using the following MRM
transitions: D0A0 at m/z 378.1 > 87.0; D0S0 at m/z 416.1 > 138.0; D0a4 at m/z 458.1 > 300.0;
D4UA-2S-GlcNCOEt-6S (HD009, Iduron Ltd) at m/z 472.0 (in source fragment ion) > 97.0 was
used as internal standard. Individual disaccharide species were identified based on their retention
times and MRM transitions using commercially-available reference standards (Iduron Ltd).
GAGs were quantified by the peak area ratio of D0A0, D0S0, and D0a4 to the internal standard
using MultiQuant 3.0.2 (Sciex). Reported GAG amounts were normalized to total protein
amounts as measured by a BCA assay (Pierce).
Heparan sulfate (HS) and dermatan sulfate (DS) calibration curves
Pure standards for D0a4 (DS/CS), D0A0 (HS), and D0S0 (HS) were dissolved in water to
generate a 1 mg/mL stock. A 14-point dilution curve in PBS was generating ranging from 0.12
ng to 1000 ng. Subsequently, the internal standard D4UA-2S-GlcNCOEt-6S (20 ng) was added
to each serial dilution. Samples were then boiled for 10 minutes at 95oC and then spun at
3,364xg to pellet any particulate matter. Supernatant was filtered using a 30kD MWCO
cellulose acetate filter plate (Millipore, MSUN03010) by spinning at 3364xg for 5 minutes at
room temperature. Resulting flow through was mixed with an equal part of acetonitrile in glass
vials and run by mass spectrometry as described above.
ELISA-based analysis of M6PR binding
The coating solution was prepared by diluting M6PR Fc to 1 µg/mL in PBS. 30 µl of coating
solution was added to each well of a 384 well ELISA plate and the plate was placed at 4oC
overnight. The following day, the plate was washed three times with wash buffer and 55 µl of
blocking buffer was added to each well. Blocking was allowed to proceed for 1 hour at room
temperature. After blocking, the plate was washed three times, and ETV:IDS or idursulfase were
added to the first column of the plate at a concentration of 100 nM. A 3-fold serial dilution was
performed across the plate, with the final column left empty as a negative control for background
binding. Each dilution series was run twice on the sample plate, and within each series every
concentration was plated in duplicate. Primary incubation of the binding reactions was done for 1
hour at room temperature. After binding, the plate was washed three time, and 25 µl of
biotinylated anti-IDS antibody diluted to 0.0625 µg/mL in sample buffer was added to each well.
The plate was incubated with detection antibody for 1 hour at room temperature after which it
was washed three times. 25 µl of streptavidin-HRP, diluted 1:50,000 in sample buffer, was then
added to each well. The plate was incubated for 30 minutes at room temperature and then
washed three times. The ELISA was developed by adding 20 µl of TMB reagent to each well,
waiting 15 minutes for development, and quenching the reaction with 40 µl of STOP solution.
The ELISA plate was read on a HighRes BioTek Synergy plate reader, where the absorbance at
450nm was recorded.
S35-sulfate accumulation assay to assess cellular potency
Healthy (GM05659, GM05565, GM03349) and MPS II patient (GM01928, GM12366,
GM13203) primary fibroblasts, all male donors aged 1-10 years, were obtained from Coriell. The
cellular S35-accumulation assay was performed using a method modified from (21). Briefly,
fibroblasts were plated at 25,000 cells/well in 96-well plates and grown in DMEM high glucose
(Gibco) with 10% FBS (Sigma). After 3 days of culture, media was replaced with low-sulfate
F12 medium (Gibco) supplemented with 10% dialyzed fetal bovine serum and 40 mCi/mL [S35]
sodium sulfate (PerkinElmer) for 96 hours. Following [S35] sodium sulfate incubation, cells were
treated with idursulfase or ETV:IDS in the presence and absence of 5 mM mannose 6-phosphate
disodium salt hydrate (Sigma) or 3 µM TV (Denali, engineered FC portion of ETV:IDS). After
24 hours of incubation, media was aspirated, cells were washed with cold PBS, and lysed with
0.01 N NaOH. Incorporated S35 was measured by scintillation counting (Microbeta Trilux). EC50
curves were generated using Prism software using a log(agonist) vs. response, variable slope
(four parameter) fit.
Animal care
All procedures in animals were performed in adherence to ethical regulations and protocols
approved by Denali Therapeutic Institutional Animal Care and Use Committee. Mice were
housed under a 12-hour light/dark cycle and had access to water and standard rodent diet
(LabDiet #25502, Irradiated) ad libitum.
Mouse strains
A previously described IDS KO mouse model on a B6N background were obtained from Jackson
Laboratories (JAX strain 024744) (33). The TfRmu/hu KI mouse line harboring the human TfR
apical domain knocked into the mouse receptor was developed by generating a knock-in (into
C57Bl6 mice) of the human apical TfR mouse line via pronuclear microinjection into single cell
embryos, followed by embryo transfer to pseudo pregnant females using CRISPR/Cas9
technology. The donor DNA comprised the human TfR apical domain coding sequence that has
been codon optimized for expression in mouse. The resulting chimeric TfR was expressed in
vivo under the control of the endogenous promoter. A founder male from the progeny of the
female that received the embryos was bred to wild-type females to generate F1 heterozygous
mice. Homozygous mice were subsequently generated from breeding of F1 generation
heterozygous mice. TfRmu/hu male mice were bred to female IDS heterozygous mice to generate
IDS KO; TfRmu/hu KI mice (method submitted for publication). All mice used in this study were
males.
Biodistribution and pharmacokinetics of ETV:IDS
For pharmacokinetic (PK) experiments, in-life blood samples were collected by submandibular
bleed at various time points. For plasma collection, blood was collected in EDTA tubes (Sarstedt
Microvette 500 K3E) and slowly inverted 10 times. Samples were centrifuged at 12,700 rpm for
7 minutes at 4°C and plasma was transferred to a fresh tube and flash-frozen on dry ice. For
serum collection, blood was allowed to clot at room temperature for at least 30 minutes. Tubes
were then centrifuged at 12,700 rpm for 7 minutes at 4°C. Serum was transferred to a fresh tube
and flash-frozen on dry ice. For biodistribution experiments, animals were deeply anesthetized
via intraperitoneal (i.p.) injection of 2.5% Avertin. Blood was collected via cardiac puncture for
serum collection. Animals were transcardially perfused with ice-cold PBS using a peristaltic
pump (Gilson Inc. Minipuls Evolution). The liver, kidney, spleen, lung, heart, and brain were
dissected and flash-frozen on dry ice.
A detailed description of parameters for each in vivo study to assess biodistribution and
pharmacokinetics can be found below.
Pharmacodynamics of ETV:IDS
For pharmacodynamic experiments, in-life serum samples were collected as described above. In-
life urine samples were collected using LabSand. Animals were individually housed in a cage
with adequate amount of LabSand to cover the bottom of the cage. During urine collection,
animals did not have access to food or water. Samples were collected at 30 minute intervals to
reduce feces contamination until an adequate amount of urine (>50 µl) was obtained. Following
collection, all urine samples were snap frozen on dry ice. For terminal sample collection, animals
were deeply anesthetized via i.p. injection of 2.5% Avertin. For CSF collection, a sagittal
incision was made at the back of the animal’s skull, subcutaneous tissue and muscle was
separated to expose the cisterna magna and a pre-pulled glass capillary tube was used to puncture
the cisterna magna to collect CSF. CSF was transferred to a Low Protein LoBind Eppendorf tube
and centrifuged at 12,700 rpm for 10 minutes at 4°C. CSF was transferred to a fresh tube and
snap frozen on dry ice. Lack of blood contamination in mouse CSF was confirmed by measuring
the absorbance of the samples at 420nm. Blood, serum and tissues were obtained ad described.
A detailed description of parameters for each in vivo study to assess pharmacodynamic
responses can be found below.
Tissue or fluid processing for downstream GAG analysis
Tissue (50mg) was homogenized in 750 µL water using the Qiagen TissueLyzer II for 3 minutes
at 30Hz. Homogenate was transferred to a 96-well deep plate and sonicated using a 96-tip
sonicator (Q Sonica) for 10x1 second pulses. Sonicated homogenates were spun at 2,500xg for
30 minutes at 4oC. The resulting lysate was transferred to a clean 96-well deep plate, and a BCA
was performed to quantify total protein amounts. 20 mg (liver) or 100 µg (brain) total protein
lysate was used for subsequent HS/DS digestion. FACS sorted cell pellets were resuspended in
digest buffer before digestion. Digestion was carried out in a PCR plate in a total volume of 100
µL (liver) or 60 µL (brain and CSF). Lysates or body fluids were mixed with Heparinases I. II,
III and Chondriotinase B in digestion buffer for 3 hours (or overnight for FACS sorted cells)
with shaking at 30oC. After the digest, EDTA and internal standard mix of HS and DS (20 ng
total) were added to each sample and the mixture was boiled at 95oC for 10 minutes. The
digested samples were spun at 3,364xg for 5 minutes and samples were transferred to a cellulose
acetate filter plate (Millipore, MSUN03010) and spun at 3,364xg for 5 minutes. The resulting
flow through was mixed with equal parts of acetonitrile in glass vials, and analyzed by mass
spectrometry as described above.
FACS-based CNS cell type isolation
To prepare a single cell suspension for sorting CNS cells, mice were perfused with PBS, whole
brains (including olfactory bulb and cerebellum) dissected, and processed into a single cell
suspension according to the manufacturers’ protocol using the adult brain dissociation kit
(Miltenyi Biotec 130-107-677). Cells were Fc blocked (Biolegend #101320, 1:100) and stained
for flow cytometric analysis with Fixable Viability Stain BV510 (BD Biosciences #564406,
1:100) to exclude dead cells, CD11b-BV421 (BD Biosciences 562605, 1:100), CD31-PerCP
Cy5.5 (BD Biosciences #562861, 1:100), O1-488 (Thermo/eBio #14-6506-82, 1:37.5), Thy1-PE
(R&D #FAB7335P, 1:100), and EAAT2-633 (Alomone #AGC-022-FR, 1:50). Cells were
washed with PBS/1% BSA and strained through a 100μm filter before sorting CD11b+
microglia, EAAT2+ astrocytes, and Thy1+ neurons on a FACS Aria III (BD Biosciences) with a
100 μm nozzle. In order to achieve pure populations of astrocytes, microglia, and neurons
negative gates were set to remove O1+ and CD31+ cells which are predominantly
oligodendrocytes and endothelial cells respectively. Sorted cells were either pelleted or collected
directly into lysis buffers, and then processed for downstream analysis including qRT-PCR,
RNA-seq, or glycomics as described in the relevant methods. Cell numbers were used to
calculate pgGAG/cell.
Distribution analysis of ETV:IDS across CNS cell types
Live cells in sheath fluid (~1.5 ml) were sorted directly into 150 uL 5% CHAPS buffer for lysis,
final concentration of CHAPS 0.5%. Samples were concentrated with Amicon Ultra 30KDa
filters to 40 uL. 5 uL of sample or recombinant ETV:IDS dilution series was run with an IgG
(human) AlphaLISA Detection Kit [PerkinElmer #AL205C] per the manufacturer’s instructions
and read on an EnVision plate reader. Sample concentrations were interpolated from the standard
curve generated using ETV:IDS and normalized to total cell input number.
Tissue processing and lipid extraction for lipidomic analysis
Frozen brain tissues (20 ± 2 mg) were transferred into 2 mL Safe-Lock Eppendorf tubes
containing a 5mm stainless steel bead and 400 µl of MS-grade methanol with internal standards.
Tissues were homogenized with Qiagen Tissuelyser for 30 sec at 25 Hz, centrifuged for 20 min
at 21,000xg at 4°C and left at -20°C for 1 hour to allow for further precipitation of proteins.
Samples were then centrifuged for 10 min at 21,000xg at 4°C. A portion of the supernatant (100
µL) was transferred into a LC-MS 96 well-plate, dried down under nitrogen steam and then
resuspended in 100 µL of acetonitrile/isopropanol/water (92.5 /5/2.5, v/v/v) with 5 mM
ammonium formate and 0.5% formic acid for glucosylceramides, galactosylceramides,
glucosylsphingosine and galactosylsphingosine analysis. The rest of the supernatant was
transferred into a separate LC-MS vials for analysis of additional lipids.
Immunohistochemistry
Fresh frozen mouse brain tissue was sectioned coronally at 10 micron thickness using a Leica
Cryostat (Leica CM 1950). Sections were directly mounted onto Fisherbrand Superfrost Plus
microscope slides and stored at -80°C until processed for immunohistochemistry. Sections were
rinsed in 1xPBS for 3 rounds of 5 minutes then fixed in 4% Paraformaldehyde for 15 minutes.
Sections were then rinsed in 1xPBS for 3 rounds of 5 minutes and incubated in Blocking
Solution (1xPBS/5% normal goat serum/0.3% Triton X-100) for 1 hour at room temperature.
Sections were then incubated in primary antibody (BioRad: Rat anti-Cd68, 1:500) prepared in
Blocking Solution for 2 hours at room temperature. Sections were rinsed in 1xPBS/0.3% Triton
X-100 for 3 rounds of 5 minutes followed by incubation in secondary antibody (Invitrogen: Goat
anti-rat Alexa Fluor 488, 1:500) and DAPI (Invitrogen Molecular Probes D1306: 1:10,000 from
5mg/mL stock) prepared in Blocking Solution for 1 hour at room temperature in the dark.
Sections were then rinsed in 1xPBS/0.3% Triton X-100 for 3 rounds of 5 minutes, quickly rinsed
in 1XPBS, and cover slipped with polyvinyl alcohol mounting medium with DABCO antifading
(Sigma 10981). Fluorescent images were taken at 20x magnification using a Zeiss Axio Scan Z1.
Each fluorophore was individually imaged with appropriate single-channel filter sets, using
identical exposure times per fluorophore across all tissue samples imaged. Individual images
were then tiled and stitched with shading correction using Zeiss Zen software.
To quantify the amount of CD68 positive cells in various brain regions across treatment groups,
immunostained coronal brain sections were fully imaged with a 20x objective in a Zeiss
AxioScan slide scanner. Images were then analyzed with Zeiss Zen 2.6. Cortical, hippocampal,
and thalamic regions of coronal brain sections were manually outlined in a blinded fashion,
based upon DAPI staining morphology. A custom-programmed analysis script was then used,
based on local dynamic thresholding and size restriction, to identify CD68-positive objects
within the selected regions. The script summed the total area of all identified CD68-positive
objects and normalized to the total tissue area analyzed for each tissue section and selected
anatomical region. A total of 3-5 mice from each treatment group were quantified.
Homogenization and Trem2 analysis of ETV:IDS Brain tissue
Tissue (50 mg) was homogenized in 500 µL 1X CST buffer (Cell Signaling Technology 9803S)
made with cOmplete Protease Inhibitor (Roche #04693132001) and PhosStop (Roche
04906837001) using the Qiagen TissueLyzer II (Cat No./ID: 85300) for 2 rounds of 3 minutes at
30 Hz. Homogenate was incubated on ice for 20 minutes and spun at 21,100 g for 30 minutes at
4oC. Subsequent lysate was transferred to a clean 96-well deep plate, and a BCA was performed
to quantify total protein amounts. Samples were then stored in the -80°C until assay use.
MSD GOLD 96w small spot streptavidin plate (MSD L45SA) was prepared for Trem2 assay by
coating with 1 µg/mL biotinylated sheep anti-mouse antibody (R&D Systems BAF1729)
overnight at 4oC. The next day, MSD plate was rinsed with tris buffered saline with triton
(TBST) and blocked for two hours using 3% bovine serum albumin in TBST, while shaking at
600 rpm. MSD plate was again rinsed again with TBST and brain lysates were diluted 5x in
blocking solution and added to the MSD plate to incubate for 1 hour at 600 rpm. Following the
next TBST rinse, sulfotagged sheep anti-mouse antibody (R&D Systems AF1729) was added to
the plate and incubated for 1 hour, again at 600 rpm, and a final rinse was conducted before
adding 2X MSD read buffer diluted in water. Plate was then read using the MSD Meso Sector
S600. Trem2 amounts were interpolated against a standard curve using recombinant Trem2, and
plotted as ng/mL.
NfL analysis of CSF
Using Quanterix Simoa Neurofilament Light (NFL) Sample Diluent (Quanterix 102252),
cerebrospinal fluid was diluted 100x before being added to Simoa 96-well microplate (Quanterix
101457). NF light assay was carried out according to Simoa NF Light Advantage Kit (Quanterix
1031086) instructions, with Simoa detector reagent and bead reagent (Quanterix 103159,
102246) being added to the samples before incubating for 30 mins, 30°C at 800rpm. Following
this, the sample plate was washed with Simoa Wash Buffer A (Quanterix 103078) on Simoa
Microplate Washer according to Quanterix two step protocol, SBG reagent (Quanterix 102250)
was added, and samples were again incubated at 30°C, 800rpm for 10 min. The two-step washer
protocol was continued, with the sample beads being twice resuspended in Simoa Wash Buffer B
(Quanterix 103079) before final aspiration of buffer. Sample NFL amounts were measured using
the Nf Light analysis protocol on the Quanterix SR-X instrument and interpolated against a
calibration curve provided with the Quanterix assay kit.
Detailed description of in vivo studies to assess biodistribution and
pharmacokinetics of ETV:IDS
PK of ETV:IDS in Wt mice (Supplementary Figure 2A)
2 month old C57Bl/6 mice (n=3 per group) were injected i.v. with a single dose of idursulfase (1
or 5.4 mg/kg body weight) or ETV:IDS (1.9 or 10 mg/kg body weight).
Biodistribution of ETV:IDS in IDS KO mice (Supplementary Figure 3C)
2-4 month old IDS KO mice (n=5 per group) were injected i.v. with a single dose 40mg/kg dose
of ETV:IDS and sacrificed at 2 hours post dose.
PK and Biodistribution of ETV:IDS in TfRmu/hu KI mice (Figure 2C and Supplementary
Figure 4)
2-3 month old TfRmu/hu mice (n=5 per group) were injected i.v. with a single dose of 50 mg/kg
body weight of IDS:Fc or ETV:IDS. Mice were sacrificed at 2 and 8 hours post dose.
PK of ETV:IDS in IDS KO; TfRmu/hu KI mice (Figure 3A)
2 month old IDS KO; TfRmu/hu KI mice were injected i.v. with idursulfase (14.2 mg/kg body
weight) or ETV:IDS (40 mg/kg body weight) either once (n=8) or once every week for 4 weeks
(n=8). For animals dosed with idursulfase or ETV:IDS, in-life serum samples were collected by
submandibular bleed at various time points.
Biodistribution of ETV:IDS in IDS KO; TfRmu/hu KI mice (Figure 3B)
2 month old IDS KO; TfRmu/hu KI mice (n=4) were injected i.v. with a single dose of idursulfase
(14.2 mg/kg body weight) or ETV:IDS (40 mg/kg body weight) and sacrificed at 2 hours post
dose.
Distribution analysis of ETV:IDS across CNS cell types in IDS KO; TfRmu/hu KI mice
(Figure 4C)
2.5 month old TfRmu/hu KI (n=4) and IDS KO; TfRmu/hu KI (n=4) mice were injected i.v. with a
single dose of vehicle or ETV:IDS (40 mg/kg body weight), respectively and sacrificed at 2
hours post dose.
Brain concentration analysis for ETV:IDS across dose levels (Figure S9)
2.5 month old IDS KO; TfRmu/hu KI mice (n=4) mice were injected i.v. with a single dose of
ETV:IDS (1, 3, or 10 mg/kg body weight), respectively and sacrificed at 2 hours post dose.
Detailed description of in vivo studies to assess pharmacodynamics of
ETV:IDS
GAG accumulation in tissues and fluids of IDS KO mice (Figure 2A)
IDS KO mice (n=5 per group) were sacrificed at 3 months of age.
Effect of peripheral administration of ETV:IDS on urine and serum in IDS KO mice
(Supplementary Figure 3A and B)
4-5 month old IDS KO mice (n=8 per group) were injected i.v. with a single dose of ETV:IDS
(1.87 mg/kg body weight). 4-5 month old littermate wild type mice (n=6), injected i.v. with
saline were used as controls.
Effect of peripheral administration of ETV:IDS on tissues in IDS KO mice (Figure 2B):
2-4 month old IDS KO mice were injected i.v. with a single dose of saline or ETV:IDS (40
mg/kg body weight) and sacrificed at 7 days (n=8 per group) post dose. Littermate wild type
mice (n=3), injected i.v. with saline were used as controls.
Effect of peripheral administration of ETV:IDS on brain and tissue GAG in IDS KO;
TfRmu/hu KI mice (Figure 3C)
2 month old IDS KO; TfRmu/hu KI mice were injected i.v. with saline, idursulfase (14.2 mg/kg
body weight), or ETV:IDS (40 mg/kg body weight) either once (n=8) or once every week for 4
weeks (n=8). 2 month-old littermate TfRmu/hu mice, injected i.v. with saline either once (n=5) or
once every week for 4 weeks (n=5) were used as controls. All animals were sacrificed either 7
days post single dose or 7 days following last 4-week dose.
GAG accumulation across CNS cell types in IDS KO; TfRmu/hu KI mice (Figure 4C)
TfRmu/hu (n=3) and IDS KO; TfRmu/hu KI mice (n=5) were sacrificed at 2 months of age.
Lysosomal lipid and RNA-seq analysis of ETV:IDS across CNS cell types (Figure 5D and
Figure 7)
3 month old IDS KO; TfRmu/hu KI mice were injected i.v. with vehicle (n=6) or ETV:IDS (10
mg/kg body weight, n=6) once every week for 4 weeks. 3 month-old littermate TfRmu/hu mice,
injected i.v. with vehicle (n=4) once every week for 4 weeks were used as controls. All animals
were sacrificed 7 days following last dose.
Effect of long-term peripheral administration of ETV:IDS on substrate accumulation,
lysosomal lipids, and neuroaxonal injury (Figure 8)
2 month old IDS KO; TfRmu/hu KI mice were injected i.v. with vehicle or ETV:IDS (1 and
3 mg/kg body weight) once every week for 13 weeks (n=10). 2 month-old littermate
TfRmu/hu mice, injected i.v. with saline once every week for 13 weeks (n=10) were used as
controls. Animals that were observed to have dose reactions, received 2 mg/kg diphenhydramine
intraperitoneally, just prior to ETV:IDS administration on subsequent dose occasions. All
animals were sacrificed 7 days following last dose.
Western blot analysis of TfR levels
Brain tissue from 3-month-old mice was collected after perfusion and 50 mg was snap frozen.
Tissue was homogenized in 500uL PBS (Gibco #10010-023), 1% NP40, with 1X protease
inhibitor (Roche #04693159001) using 5mm beads in a tissue lyser block for 6 minutes at 30Hz.
Samples were then solubilized for 10 minutes on ice before sedimentation at 18.8K G for 20
minutes at 4’C. Lysate was prepared for SDS-PAGE by diluting with 4X NuPage Sample Buffer
(Invitrogen #NP007), 10X NuPage Reducing Buffer (Invitrogen #NP009), and RIPA Buffer
(Teknova #R3792). Samples were boiled for 5 minutes, and then run on a 4-12% Bis-Tris gel
(Invitrogen #NP0322) in MOPS buffer (Invitrogen #NP000102), 120V for 1.5 hours. Gel was
then transferred on the bio-rad semi-dry transfer apparatus high molecule weight program onto
nitrocellulose. Transferred blot was blocked with Rockland Blocking buffer (Rockland #MB-
070). Blot was then probed for Transferrin receptor (Thermofisher #13-6890, 1:1000) and
GAPDH (Sigma #G8795, 1:2500) and detected via fluorescent secondaries (Licor) while
incubating in Rockland Blocking Buffer. After washing, blot was imaged and quantified on the
Odyssey scanner, and analyzed in PRISM by on way ANOVA, Tukeys post hoc.
GAG analysis of ETV:IDS across CNS cell types (Figure 4E)
3.5 month old IDS KO; TfRmu/hu KI mice were injected i.v. with Vehicle (n=6) or ETV:IDS (40
mg/kg body weight, n=6) once every week for 4 weeks. 3.5 month-old littermate TfRmu/hu mice,
injected i.v. with Vehicle (n=4) once every week for 4 weeks were used as controls. All animals
were sacrificed 7 days following last 4-week dose.
Mass spectrometry imaging of ganglioside levels in brain tissue following peripheral
administration of ETV:IDS in IDS KO; TfRmu/hu KI mice (Figure 5E)
2.5 month old IDS KO; TfRmu/hu KI mice were injected i.v. with Vehicle, idursulfase (18.4 mg/kg
body weight, activity-equivalent dose of ETV:IDS), or ETV:IDS (40 mg/kg body weight) once
every week for 4 weeks (n=3). 2.5 month-old littermate TfRmu/hu mice, injected i.v. with Vehicle
once every week for 4 weeks (n=3) were used as controls. All animals were sacrificed 7 days
following last dose.
RNA-seq analysis of gene expression isolated CNS cell types
Live cells were sorted directly in 350uL RLT-plus buffer (Qiagen) with 1:100 beta-
mercaptoethanol. RNA was extracted using the RNeasy Plus Micro Kit (Qiagen, 74034) and
resuspended in 14μL nucleasefree water. RNA quantity and quality were assessed with a RNA
6000 Pico chip (Agilent 5067-1513) on a 2100 Bioanalyzer (Agilent).
Figure 4, SI 7: For QuantSeq library prep, RNA was processed using
the QuantSeq 3' mRNAseq Library Prep Kit FWD for Illumina (Lexogen) with the UMI second
strand synthesis module in order to identify and remove PCR duplicates, following the ‘low-
input’ protocol defined by the manufacturer. Barcoded samples were quantified using
the NEBNext Library Quant Kit for Illumina (NEB, E7630S). All samples were pooled in
equimolar ratios into one sequencing library, which was quantified on a Bioanalyzer with a High
Sensitivity DNA chip (Agilent, 5067-4626). 65 bp single end reads were generated on an
Illumina HiSeq 4000 lane at the UCSF Center for Advanced Technology.
Figure 7, Fig S7: For Illumina Next Generation Sequencing, samples were processed using the
Clontech SMART-Seq v4 3′ DE (Takara Bio USA, Inc. 635040 ) kit for cDNA synthesis and the
Nextera XT DNA Library Prep Kit (Illumina FC-131-1096) for library generation. Up to 1 ng
of total RNA per sample was used for reverse transcription followed by 13 cycles of cDNA
amplification to produce full length cDNA. After purification, cDNA quantification was
performed using Bioanalyzer High Sense DNA chip (Agilent 5067-4626) and normalized to 300
pg of input for library preparation. The tagmentation step simultaneously fragmented and
introduced adaptors to the DNA strands, followed by 12 cycles of PCR amplification. Library
quantity and quality were assessed with Qubit 1X dsDNA HS Assay Kits (Invitrogen Q33231)
and Bioanalyzer High Sense DNA chip. Libraries were pooled in equimolar ratios for
sequencing on two Illumina NextSeq flowcells at SeqMatic, 75 bases single end reads.
Raw data processing
Figure 4: UMIs were extracted from raw sequencing reads using umi2index (Lexogen) and
sequencing adapters were trimmed with skewer (76). Reads were aligned to the mouse genome
version GRCm38_p6. A STAR index (version 2.5.3a;(77)) was built with the –
sjdbOverhang=50 argument. Splice junctions from Gencode gene models (release M17) were
provided via the –sjdbGTFfile argument. STAR alignments were generated with the following
parameters: –outFilterType BySJout, –quantMode TranscriptomeSAM, –outFilterIntronMotifs
RemoveNoncanonicalUnannotated, –outSAMstrandField intronMotif, –outSAMattributes NH
HI AS nM MD XS and –outSAMunmapped Within. Alignments were obtained with the
following parameters: –readFilesCommand zcat –outFilterType BySJout –
outFilterMultimapNmax 20 –alignSJoverhangMin 8 –alignSJDBoverhangMin 1 –
outFilterMismatchNmax 999 –outFilterMismatchNoverLmax 0.6 –alignIntronMin 20 –
alignIntronMax 1000000 –alignMatesGapMax 1000000 –quantMode GeneCounts –
outSAMunmapped Within –outSAMattributes NH HI AS nM MD XS –outSAMstrandField
intronMotif –outSAMtype BAM SortedByCoordinate –outBAMcompression 6. Alignments
mapped to the same genomic location that shared the same UMI were collapsed using the
collapse_UMI_bam tool (Lexogen). Gene level counts were obtained using feature Counts from
the subread package (version 1.6.2; (78)). Gene s; ymbols and biotype information were
extracted from the Gencode GTF file.
Figure S7: Sequencing adapters were trimmed from the raw reads with skewer(76). Reads were
aligned to the mouse genome version GRCm38_p6. A STAR index (version 2.7.1a; (77)) was
built with the –sjdbOverhang=50 argument. Splice junctions from Gencode gene models (release
M17) were provided via the –sjdbGTFfile argument. STAR alignments were generated with the
following parameters: –outFilterType BySJout, –quantMode TranscriptomeSAM, –
outFilterIntronMotifs RemoveNoncanonicalUnannotated, –outSAMstrandField intronMotif, –
outSAMattributes NH HI AS nM MD XS and –outSAMunmapped Within. Alignments were
obtained with the following parameters: –readFilesCommand zcat –outFilterType BySJout –
outFilterMultimapNmax 20 –alignSJoverhangMin 8 –alignSJDBoverhangMin 1 –
outFilterMismatchNmax 999 –outFilterMismatchNoverLmax 0.6 –alignIntronMin 20 –
alignIntronMax 1000000 –alignMatesGapMax 1000000 –quantMode GeneCounts –
outSAMunmapped Within –outSAMattributes NH HI AS nM MD XS –outSAMstrandField
intronMotif –outSAMtype BAM SortedByCoordinate –outBAMcompression 6. Gene level
counts were obtained using feature Counts from the subread package (version 1.6.2; (78)). Gene
symbols and biotype information were extracted from the Gencode GTF file.
Gene Expression Analysis
Figures 4, 7, S 7: RNA-seq expression analyses were performed with R Figure 1 (R Core Team
2018; version 3.2) and Figure 5 (R Core Team 2019; version 3.6), using the voom analysis
framework (79) from the limma package (80). Gene expression profiles were TMM normalized
(81) and low abundance genes were identified and removed prior to downstream analysis. Low
abundance genes were defined as those which were not expressed higher than 10 ten counts per
million (CPM) in at least four samples.
Principal components analysis
Log-transformed CPM expression values from the top 500 genes with the highest variance were
used for principal components analysis (PCA).
Enrichment score of published cell type markers
To visualize the enrichment / depletion of commonly used marker transcripts across the FACS
sorted samples, normalized counts per million (CPM) for the following curated cell type marker
genes were extracted from the dataset:
Astrocytes: Aqp4, Cyp4f15, Fgfr3, Gfap, Slc1a3
Endothelial cells: Cldn5, Esam, Pecam1
Microglia: Ccl3, P2ry13, Tmem119, Tnf, Trem2
Neurons: Map1b, Slc17a7, Snap25, Syt1, Tubb3
Oligodendrocytes: Mbp, Mog, Plp1
After addition of five pseudocounts, CPMs were log2 transformed and for each gene the mean
value observed in the input samples was subtracted, centering the scores on the baseline
expression observed in the unsorted samples. The resulting expression matrix was visualized as a
heatmap with the ComplexHeatmap R package(82) with colors showing log2 fold enrichment /
depletion relative to the mean expression of the gene in the unsorted samples.
To summarize marker gene expression in each sample as a single score for each cell type, we
calculated a weighted mean across all markers for a cell type in each sample. Weights were
determined by the loading of each marker gene on the first eigengene of the log2-transformed
centered expression matrix(83).
To visualize the enrichment / depletion of commonly used marker transcripts across the FACS
sorted samples, normalized counts per million (CPM) for the following curated cell type marker
genes were extracted from the dataset following the cell type specific gene set markers identified
by Zhang et al 2014(38). After addition of five pseudocounts, CPMs were log2 transformed and
for each gene the mean value observed in the input samples was subtracted, centering the scores
on the baseline expression observed in the unsorted samples. The resulting expression matrix
was visualized as a heatmap with the ComplexHeatmap R package with colors showing log2 fold
enrichment / depletion relative to the mean expression of the gene in the unsorted samples. To
summarize marker gene expression in each sample as a single score for each cell type, we
calculated a weighted mean across all markers for a cell type in each sample. Weights were
determined by the loading of each marker gene on the first eigengene of the log2-transformed
centered expression matrix(83).
Differential gene expression analysis & Gene set enrichment analysis
To identify differentially expressed genes, each sample was annotated with an experimental
"group" based on the genotype (TfRmu/hu / IDS KO; TfRmu/hu KI), treatment (ETV:IDS / vehicle)
and FACS enriched cell type (unsorted / astrocytes / microglia / neurons). Normalized counts
were log2 transformed and sample- and observation-weights were determined using voom(81).
Afterward, a linear model including the "group" and "sex" covariates was fit across all samples
using the limma R package(80). The following contrasts were extracted from the fit, separately
for each cell type:
1. IDS KO; TfRmu/hu KI (vehicle) compared to TfRmu/hu KI (vehicle)
2. IDS KO; TfRmu/hu KI (ETV:IDS) compared to IDS KO; TfRmu/hu KI (vehicle)
and empirical Bayes statistics for differential expression were calculated. P-values were
corrected for multiple testing by calculating the local false discovery rate (FDR) according to
Benjamini & Hochberg(84). Results were displayed as volcano plots, displaying the negative
decadic logarithm of the FDR on the y-axis and the log2 fold difference on the x-axis. Genes
passing the following thresholds: nominal P-value < 0.1 and absolute log2 fold change > 1 were
highlighted. The normalized expression (CPM) across all samples was also visualized as
boxplots for the top six most significantly up- or down-regulated genes (by P-value) between
IDS KO; TfRmu/hu KI (vehicle) and TfRmu/hu (vehicle) conditions, separately for each cell type.
Differential expression analysis of "DAM signature" and "CLEAR network" genes was
performed at the gene-set level with the camera algorithm(85) on the normalized, voom-
transformed expression scores, using the default value for inter-gene correlations within the gene
sets (0.01). The following contrast was evaluated: " TfRmu/hu;IDS KO (vehicle) compared to
TfRmu/hu;IDS KO (ETV:IDS)".
DAM signature genes
To examine the expression of "Disease associated microglia (DAM)" genes(46), we retrieved the
top 50 genes identified by Keren-Shaul et al as up-regulated in DAM versus homeostatic
microglia in 5xFAD animals from Supplemental Table S2: Actr3b, Ank, Apoe, Axl, B2m,
Baiap2l2, Capg, Ccl3, Ccl4, Ccl6, Cd52, Cd63, Cd9, Ch25h, Clec7a, Cox6a2, Csf1, Cst7, Ctsb,
Ctsd, Ctsl, Ctsz, Cxcl14, Dkk2, Fabp3, Fam20c, Flt1, Fth1, Gm1673, Gnas, Gusb, H2-D1, Hpse,
Igf1, Itgax, Lgals3bp, Lgi2, Lox, Lpl, Lyz1, Lyz2, Mamdc2, Mif, Nceh1, Psat1, Rps2, Serpine2,
Spp1, Sulf2, Tyrobp. The normalized log2 expression scores (CPMs) of these genes in microglia,
centered on the baseline expression in the unsorted samples, were visualized as a heatmap with
the ComplexHeatmap R package(82). Rows (genes) were clustered based on euclidean distance
with the "ward.D" clustering method.
CLEAR network genes
To examine the expression of "CLEAR network" genes, we curated the following gene symbols
from the publication by Palmieri et al, 2011(47): Asah1, Atp6ap1, Atp6v0a1, Atp6v0b, Atp6v0c,
Atp6v0d1, Atp6v0d2, Atp6v0e, Atp6v1a, Atp6v1b2, Atp6v1c1, Atp6v1d, Atp6v1e1, Atp6v1g1,
Atp6v1h, Becn1, Bloc1s1, Bloc1s3, Cd63, Clcn7, Cln3, Ctns, Ctsa, Ctsb, Ctsd, Ctsf, Gaa,
Gabarap, Galns, Gba, Gla, Glb1, Gnptg, Gns, Gusb, Hexa, hexb, Hif1a, Hps1, Hps3, Hps5,
Igf2r, Lamp1, M6pr, Mcoln1, Naglu, Nagpa, Neu1, Nrbf2, Plbd2, Ppt1, Prkag2, Psap, Rab7,
Rragc, Scpep1, Sgsh, Slc36a1, Sqstm1, Stk4, Sumf1, Tpp1, Uvrag, Vps11, Vps18, Vps26a,
Vps33a, Vps35, Vps8. The normalized log2 expression scores (CPMs) of these genes in
microglia, centered on the baseline expression in the unsorted samples, were visualized as a
heatmap with the ComplexHeatmap R package(82) . Rows (genes) were clustered based on
euclidean distance with the "ward.D" clustering method.
LC-MS assay for BMP and gangliosides
BMP and gangliosides analyses were performed by liquid chromatography (Shimadzu Nexera
X2 system, Shimadzu Scientific Instrument) coupled to electrospray mass spectrometry (Sciex
QTRAP 6500+, Sciex). For each analysis, 5 µL of sample was injected on a BEH C18 1.7 µm,
2.1×100 mm column (Waters Corporation) using a flow rate of 0.25 mL/min at 55°C. Mobile
phase A consisted of 60:40 acetonitrile/water (v/v) with 10 mM ammonium acetate Mobile phase
B consisted of 90:10 isopropyl alcohol /acetonitrile (v/v) with 10 mM ammonium acetate. The
gradient was programmed as follows: 0.0–0.01 min from 45% B to 99% B, 0.1–3.0 min at 99%
B, 3.0–3.01 min to 45% B, and 3.01–3.50 min at 45% B. Electrospray ionization was performed
in negative ion mode applying the following settings: curtain gas at 30; collision gas was set at
medium; ion spray voltage at -4500; temperature at 600°C; ion source Gas 1 at 50; ion source
Gas 2 at 60. Data acquisition was performed using Analyst 1.6.3 (Sciex) in multiple reaction
monitoring mode (MRM), with the following parameters: dwell time (msec) for each species
reported in the Table S1, entrance potential (EP) at -10; and collision cell exit (CXP) potential at
-15. BMP and gangliosides species were quantified using the non-endogenous internal standards
BMP di14:0 and GM3 (d18:1/18:0(d5)). Quantification was performed using MultiQuant 3.02
(Sciex). BMP and gangliosides concentration were normalized to either total protein amount,
tissue weight or volume. Protein concentration was measured using the bicinchoninic acid
(BCA) assay (Pierce).
LC-MS assay for GlcCer and GalCer
Glucosylceramide and galactosylceramide analyses were performed by liquid chromatography
(Shimadzu Nexera X2 system, Shimadzu Scientific Instrument) coupled to electrospray mass
spectrometry (Sciex QTRAP 6500+ Sciex). For each analysis, 10 µL of sample was injected on a
HALO HILIC 2.0 µm, 3.0 × 150 mm column (Advanced Materials Technology, PN 91813-701)
using a flow rate of 0.45 mL/min at 45°C. Mobile phase A consisted of 92.5/5/2.5
ACN/IPA/H2O with 5 mM ammonium formate and 0.5% formic Acid. Mobile phase B
consisted of 92.5/5/2.5 H2O/IPA/ACN with 5 mM ammonium formate and 0.5% formic Acid.
The gradient was programmed as follows: 0.0–3.1 min at 100% B, 3.2 min at 95% B, 5.7 min at
85% B, hold to 7.1 min at 85% B, drop to 0% B at 7.25min and hold to 8.75 min, ramp back to
100% at 10.65 min and hold to 11 min. Electrospray ionization was performed in the positive-ion
mode applying the following settings: curtain gas at 25; collision gas was set at medium; ion
spray voltage at 5500; temperature at 350°C; ion source Gas 1 at 55; ion source Gas 2 at 60. Data
acquisition was performed using Analyst 1.6 (Sciex) in multiple reaction monitoring mode
(MRM) with the following parameters: dwell time (msec) and collision energy (CE) for each
species reported in Table S2; entrance potential (EP) at 10; and collision cell exit potential
(CXP) at 12.5. Lipids were quantified using a mixture of isotope labeled internal standards as
reported in Table S2. Quantification was performed using MultiQuant 3.02 (Sciex).
LC-MS analysis of Eicosanoids
Eicosanoid analyses were performed by liquid chromatography (Shimadzu Nexera X2 system,
Shimadzu Scientific Instrument) coupled to electrospray mass spectrometry (Sciex QTRAP
6500+, Sciex). For each analysis, 5 µL of sample was injected on a BEH C18 1.7 µm, 2.1×100
mm column (Waters Corporation) using a flow rate of 0.6 mL/min at 40°C. Mobile phases were
composed as follows: A = water + 0.1% acetic acid, and B = 90∶10 acetonitrile/isopropyl
alcohol (v/v). The gradient was programmed as follows: 0.0–1.0 min at 25% B; 1.0–8.5 min to
95% B; 8.50–8.51 min at 95%B; 8.51–10.00 min at 25% B. Electrospray ionization was
performed in negative ion mode applying the following settings: curtain gas at 30; collision gas
was set at medium; ion spray voltage at -4500; temperature at 600°C; ion source Gas 1 at 50; ion
source Gas 2 at 60. Data acquisition was performed using Analyst 1.6.3 (Sciex) in multiple
reaction monitoring mode (MRM), with the following parameters: dwell time (msec), collision
energy (CE), and declustering potential (DP) for each species reported in Table S3; entrance
potential (EP) at -10; and collision cell exit potential (CXP) at -12. Eicosanoids were quantified
using a mixture of non endogenous, deuterated internal standards as reported in Table S3.
Quantification was performed using MultiQuant 3.02 (Sciex).
Lipidomics analysis
Lipid analyses were performed by liquid chromatography (Shimadzu Nexera X2 system,
Shimadzu Scientific Instrument) coupled to electrospray mass spectrometry (QTRAP
6500+, Sciex). For each analysis, 5 µL of sample was injected on a BEH C18 1.7 µm, 2.1×100
mm column (Waters Corporation) using a flow rate of 0.25 mL/min at 55°C. For positive
ionization mode, mobile phase A consisted of 60:40 acetonitrile/water (v/v) with 10 mM
ammonium formate with 0.1% formic acid; mobile phase B consisted of 90:10 isopropyl
alcohol/acetonitrile (v/v) with 10 mM ammonium formate with 0.1% formic acid. For negative
ionization mode, mobile phase A consisted of 60:40 acetonitrile/water (v/v) with 10 mM
ammonium acetate; mobile phase B consisted of 90:10 isopropyl alcohol/acetonitrile (v/v) with
10 mM ammonium acetate. The gradient was programmed as follows: 0.0–8.0 min from 45% B
to 99% B, 8.0–9.0 min at 99% B, 9.0–9.1 min to 45% B, and 9.1–10.0 min at 45% B.
Electrospray ionization was performed in either positive or negative ion mode applying the
following settings: curtain gas at 30; collision gas was set at medium; ion spray voltage at 5500
(positive mode) or 4500 (negative mode); temperature at 250°C (positive mode) or 600°C
(negative mode); ion source Gas 1 at 50; ion source Gas 2 at 60. Data acquisition was performed
using Analyst 1.6.3 (Sciex) in multiple reaction monitoring mode (MRM), with the following
parameters: dwell time (msec) and collision energy (CE) for each species reported in
Table S4 (negative mode) or Table S5 (positive mode); DP at 80 (positive mode) and at -80
(negative mode); EP at 10 (positive mode) or -10 (negative mode); and CXP at 12.5 (positive
mode) or -12.5 (negative mode). Lipids were quantified using a mixture of non-endogenous
internal standards as reported in Tables S4 and S5. Quantification was performed
using MultiQuant 3.02 (Sciex).
Mass spectrometry imaging of ganglioside levels
Sample preparation
Brain tissue was flash frozen on aluminum foil that was slowly lowered into liquid nitrogen for
approximately 10 seconds. Frozen brains were stored at -80°C until ready for use. Prior to
sectioning, the brains were removed from the -80°C freezer and placed in the cryostat chamber to
equilibrate to -20°C. Brains were cut on a cryostat (Leica Biosystems) into 12 µm thick sections
and thaw-mounted onto indium-tix oxide (ITO) coated slides (Delta Technologies). Two brain
levels were collected at approximately +0.72mm and –1.82mm from Bregma. Plates with
sections designated for MSI were washed with 50 mM ammonium formate (chilled to 4°C), three
times, for 10 seconds each. The plates were allowed to dry at room temperature prior to matrix
application. Additional sections were obtained for H&E staining. After staining, digital
micrographs were obtained via a slide scanner (Leica Biosystems).
Matrix application
The plates were coated with 1,5-diaminonaphthalene (DAN) MALDI matrix via sublimation(86,
87). Briefly, 100 mg of recrystallized DAN was placed in the bottom of a glass sublimation
apparatus (Chemglass Life Sciences). The apparatus was placed on a metal heating block set to
130°C and DAN was sublimated onto the tissue surface for 4 minutes at a pressure of less than
25 mTorr. Approximately 1.8 mg of DAN was applied to each ITO slide, determined by
weighing the slide before and after matrix application. The coated plates were then placed in a
Petri dish, flushed with nitrogen gas, and stored at -80°C for two days prior to MS analysis(88).
Mass spectrometry imaging
The plates were removed from the freezer and allowed to equilibrate to room temperature prior
to removing them from their sealed Petri dish. The brain sections were imaged on a Solarix 15T
FT-ICR MS (Bruker Daltonics), equipped with a SmartBeam II 2 kHz frequency tripled
Nd:YAG laser (355 nm). Images were acquired at 100 µm spatial resolution in negative ion
mode. Each pixel is the average of 1000 laser shots using the small laser focus setting and
random-walking within the 100 µm pixel. The mass spectrometer was externally calibrated with
a series of phosphorus clusters. Data were collected from m/z 600 – 3,000 with a time-domain
file size of 1 M (FID length = 1.3631 sec), resulting in a resolving power of 153,000 at m/z 1041.
Images were generated using FlexImaging 3.0 (Bruker Daltonics). Gangliosides were identified
by accurate mass, with the mass accuracies typically better than 1 ppm.
Fig. S1. Validation of LC-MS/MS methods and cellular experimental models.
Specific activity of IDS was assessed in IDS KO cell lines (A) and MPS II patient fibroblast cells
(B) and normalized to the activity measured in their respective control lines; n=3 experiments in
(A) and n=2-3 experiments in (B). Graphs display mean SEM. C) The most abundant heparan
sulfate (D0S0, D0A0) and dermatan sulfate (D0a4) disaccharides were identified in IDS KO cell
lysates and peaks verified by purified standards; representative traces shown. D) Standard curves
were generated using the ratio of peak area to internal standard peak area and fitted to a second-
order polynomial with a weighted sum of squares (1/Y2). Accuracy test of interpolated standards
(right) determines the dynamic range of the assay; n=3 mean SD. (E-F) The LC-MS/MS assay
was applied to HEK293T (E) and MPS II patient fibroblasts (F) to quantify accumulated D0S0,
D0A0, and D0a4 in IDS-deficient cells; n=6 experiments with 3 lines per genotype in (E) and
n=3 experiments with 3 lines per phenotype in (F). Graphs display mean SD with p values:
multiple t-test analysis with Holm-Sidak multiple comparison test; ** P < 0.01, *** P < 0.001,
and **** P < 0.0001.
Fig. S2. PK of ETV:IDS in wild-type mice.
Plasma concentrations of IDS in wild-type mice following a single, intravenous injection of 17
or 90 nmol/kg of ETV:IDS or idursulfase; n=3. Graphs display mean SD with summary table
of mean clearance and terminal half-life values.
Fig. S3. GAG reduction in fluids and peripheral tissue biodistribution of ETV:IDS in IDS
KO mice. Urine (A) and serum (B) GAG levels in IDS KO mice were assessed before and after
administration of a single intravenous injection of ETV:IDS and compared to wild-type mice;
n=8 for serum and 6 for urine. C) Liver, spleen, and lung concentrations of IDS in IDS KO mice
were measured two hours after a single intravenous injection of 40 mg/kg ETV:IDS; n=5 per
group. Graphs display mean SEM.
Fig. S4. PK of ETV:IDS in TfRmu/hu KI mice.
Serum concentrations of ETV:IDS or Fc:IDS in TfRmu/hu KI mice following a single intravenous
injection of 50 mg/kg dose; n=4-5, graphs display mean SD with summary table of clearance
values.
Fig. S5. IDS deletion does not affect TfR levels in the brain. (A) The levels of TfR were
assessed in brains of TfRmu/hu KI and IDS KO; TfRmu/hu KI dosed with vehicle or ETV:IDS by
western blot analysis. (B) Quantification of TfR levels across groups; n=5 for TfRmu/hu KI group
and n=8 for IDS KO; TfRmu/hu KI mice groups. Graphs display mean SEM.
Fig. S6. Individual disaccharide analysis from the brain and CSF of dosed IDS KO;
TfRmu/hu KI mice. Individual disaccharides derived from heparan sulfate (D0S0 and D0A0) and
dermatan sulfate (D0a4) were measured in brain (A) and CSF (B) from IDS KO; TfRmu/hu KI
mice administered a single intravenous injection of 40 mg/kg ETV:IDS or 14.2 mg/kg
idursulfase (activity-equivalent dose) or four, weekly doses of enzyme. n=8 per IDS KO;
TfRmu/hu KI groups and n=5 for the TfRmu/hu KI group. Graphs display mean ± SEM and p
values: one-way ANOVA with Dunnett multiple comparison test; ** P < 0.01, *** P < 0.001,
and **** P < 0.0001.
Fig. S7. Characterization of FACS-isolated CNS cell types. (A) Representative FACS gates
used to isolate enriched populations of cells, from top left to bottom right: forward (FSC) and
side (SSC) scatter determines cells from debris, live cells are positively gated, exclusion of CD31
positive endothelial cells is confirmed, and removal of O1 oligodendrocytes cells in CD11B
microglia, EAAT2 astrocytes, and Thy1 neurons cell populations determines final sort criteria.
(B) Cell populations from 4 WT mice isolated by the FACS protocol cluster together by
principle component analysis for sample type and biological replicate. PC1 and PC2 account for
47.68% and 25.17% of the variance, respectively. (C) Boxplot of weighted mean marker gene
expression of classic cell-specific markers identified from the literature compared to unsorted
cells (Input, 100%) demonstrating enrichment and depletion scores of cell type specific marker
genes in each population isolated via FACS. (D) Log2FC of weighted mean for all genes
identified as cell-type specific gene marker sets(38).
Fig. S8. Analysis of ganglioside, BMP, and glucosylceramide levels in FACS-isolated CNS
cell types after treatment with ETV:IDS. The summed levels of ganglioside (A), BMP (B),
and ganglioside (C) species are shown from isolated neurons, astrocytes, and microglia from IDS
KO; TfRmu/hu KI mice after four, weekly doses of 10mg/kg ETV:IDS, compared to vehicle
treatment and TfRmu/hu KI mice; n=6 per IDS KO; TfRmu/hu KI group and n=4 for TfRmu/hu KI
group. Graphs display mean ± SEM and P values: one-way ANOVA with Tukey multiple
comparison test; * P < 0.05, *** P < 0.001, and **** P < 0.0001.
Fig. S9. Relationship between dose level and brain concentration of ETV:IDS. IDS KO;
TfRmu/hu KI mice were administered a single, intravenous injections of 1, 3, or 10 mg/kg
ETV:IDS as described, and the levels of ETV:IDS were measured in brain two hours post-dose.
n=4 mice per dose level. Graphs display mean ± SEM and P values: one-way ANOVA with
Dunnett multiple comparison test; * P < 0.05, ** P < 0.01, and *** P < 0.001.
Fig. S10. Analysis of lysosomal lipids in CSF after chronic treatment with ETV:IDS. IDS
KO; TfRmu/hu KI mice were administered thirteen weekly, intravenous injections of 1 or 3 mg/kg
ETV:IDS as described. Glucosylceramide, BMP, and GM3 ganglioside levels were measured in
CSF of IDS KO; TfRmu/hu KI and TfRmu/hu KI mice following chronic dosing; n=9-10 per IDS
KO; TfRmu/hu KI groups and n=10 for TfRmu/hu KI group. Graphs display mean ± SEM and p
values: one-way ANOVA with Dunnett multiple comparison test; * P < 0.05, *** P < 0.001, and
**** P < 0.0001.
Table S1. LC-MS acquisition parameters information for the BMP and gangliosides assay.
Lipid INTERNAL STANDARD Q1 (m/z) Q3 (m/z)
DP
(V)
CE
(V)
BMP(14:0/14:0) INTERNAL STANDARD 665.3 227.2 -80 -50
BMP(20:4/20:4) BMP(14:0/14:0) 817.5 303.3 -80 -50
BMP(22:6/22:6) BMP(14:0/14:0) 865.5 327.3 -80 -50
BMP(18:1/18:1) BMP(14:0/14:0) 773.5 281.3 -80 -50
GM3(d18:1/18:0(d5)) INTERNAL STANDARD 1184.8 290.1 -60 -65
GM3(d34:1) GM3(d18:1/18:0(d5)) 1151.7 290.1 -60 -65
GM3(d36:1) GM3(d18:1/18:0(d5)) 1179.8 290.1 -60 -65
GM3(d38:1) GM3(d18:1/18:0(d5)) 1207.8 290.1 -60 -65
GM3(d40:1) GM3(d18:1/18:0(d5)) 1235.8 290.1 -60 -65
GD3(d34:1) GM3(d18:1/18:0(d5)) 720.9 290.1 -60 -40
GD3(d36:1) GM3(d18:1/18:0(d5)) 734.9 290.1 -60 -40
GD3(d38:1) GM3(d18:1/18:0(d5)) 748.9 290.1 -60 -40
GD3(d40:1) GM3(d18:1/18:0(d5)) 762.9 290.1 -60 -40
GD3(d42:2) GM3(d18:1/18:0(d5)) 775.9 290.1 -60 -40
GD3(d42:1) GM3(d18:1/18:0(d5)) 776 290.1 -60 -40
GD1a/b(d36:1) GM3(d18:1/18:0(d5)) 917.5 290.1 -60 -52
GD1a/b(d38:1) GM3(d18:1/18:0(d5)) 931.5 290.1 -60 -52
GT1b(d36:1) GM3(d18:1/18:0(d5)) 1063 290.1 -60 -35
GT1b(d38:1) GM3(d18:1/18:0(d5)) 1077 290.1 -60 -35
GQ1b(d36:1) GM3(d18:1/18:0(d5)) 1208.6 290.1 -60 -55
GQ1b(d38:1) GM3(d18:1/18:0(d5)) 1222.6 290.1 -60 -55
Table S2. LC-MS acquisition parameters information for GlcCer and GalCer assay.
Lipid INTERNAL
STANDARD Q1 (m/z) Q3 (m/z) DP (V) CE (V)
GlcCer(d18:1/16:0) GlcCer(d18:1(d5)/18:0) 700.6 264.3 45 45
GlcCer(d18:1/18:0) GlcCer(d18:1(d5)/18:0) 728.6 264.3 45 45
GlcCer(d18:2/18:0) GlcCer(d18:1(d5)/18:0) 726.6 262.3 45 45
GlcCer(d18:1/20:0) GlcCer(d18:1(d5)/18:0) 756.6 264.3 45 50
GlcCer(d18:2/20:0) GlcCer(d18:1(d5)/18:0) 754.6 262.3 45 50
GlcCer(d18:1/22:0) GlcCer(d18:1(d5)/18:0) 784.7 264.3 45 50
GlcCer(d18:1/22:1) GlcCer(d18:1(d5)/18:0) 782.7 264.3 45 50
GlcCer(d18:2/22:0) GlcCer(d18:1(d5)/18:0) 782.7 262.3 45 50
GlcCer(d18:1/24:1) GlcCer(d18:1(d5)/18:0) 810.7 264.3 45 50
GlcCer(d18:1/24:0) GlcCer(d18:1(d5)/18:0) 812.7 264.3 45 50
Glucosyl sphingosine Glucosyl sphingosine
(d5)
462.2 267.3 45 16
GlcCer(d18:1
(d5)/18:0)
INTERNAL
STANDARD
733.6 269.3 45 45
Glc Sphingosine(d5) INTERNAL
STANDARD
467.2 269.3 30 16
GalCer(d18:1/16:0) GlcCer(d18:1(d5)/18:0) 700.6 264.3 45 45
GalCer(d18:1/18:0) GlcCer(d18:1(d5)/18:0) 728.6 264.3 45 45
GalCer(d18:2/18:0) GlcCer(d18:1(d5)/18:0) 726.6 262.3 45 50
GalCer(d18:1/20:0) GlcCer(d18:1(d5)/18:0) 756.6 264.3 45 50
GalCer(d18:2/20:0) GlcCer(d18:1(d5)/18:0) 754.6 262.3 45 50
GalCer(d18:1/22:0) GlcCer(d18:1(d5)/18:0) 784.7 264.3 45 50
GalCer(d18:1/22:1) GlcCer(d18:1(d5)/18:0) 782.7 264.3 45 50
GalCer(d18:2/22:0) GlcCer(d18:1(d5)/18:0) 782.7 262.3 45 50
GalCer(d18:1/24:1) GlcCer(d18:1(d5)/18:0) 810.7 264.3 45 50
GalCer(d18:1/24:0) GlcCer(d18:1(d5)/18:0) 812.7 264.3 45 50
Galactosyl sphingosine Glucosyl
sphingosine(d5)
462.2 264.3 45 16
alpha-GalCer
(d18:1/16:0)
GlcCer(d18:1(d5)/18:0) 700.6 264.3 45 45
Table S3. LC-MS acquisition parameters information for eicosanoid assay.
Lipid
INTERNAL STANDARD Q1 (m/z) Q3 (m/z) DP (V) CE (V)
12-HETE-d8 INTERNAL STANDARD 327.3 184.1 -65 -20
15-HETE-d8 INTERNAL STANDARD 327.3 226.1 -65 -20
5-HETE-d8 INTERNAL STANDARD 327.3 116.1 -65 -20
PGE2-d4 INTERNAL STANDARD 355.31 275.1 -35 -23
PGF2alpha-d4 INTERNAL STANDARD 357.2 197.1 -60 -35
6-k-PGF1alpha-d4 INTERNAL STANDARD 373.2 249.1 -45 -35
Arachidonic Acid-d8 INTERNAL STANDARD 311.2 267.1 -110 -18
5-HETE 5-HETE-d8 319.2 115.1 -50 -18
12-HETE 12-HETE-d8 319.2 179.1 -50 -18
15-HETE 15-HETE-d8 319.2 219.1 -50 -18
6k-PGF1alpha 6-k-PGF1alpha-d4 369.2 245.1 -45 -34
TXB2 15-HETE-d8 369.2 169.1 -50 -22
PGF2alpha PGF2alpha-d4 353.2 193.1 -50 -35
PGE2 PGE2-d4 351.2 271.1 -30 -28
5,15-diHETE 15-HETE-d8 335.2 115.1 -35 -18
5-iso-PGF2alpha PGF2alpha-d4 353.2 115.1 -60 -28
PGD2 PGE2-d4 351.2 233.2 -30 -28
9-HOTrE 15-HETE-d8 293 171 -45 -15
9-OxoODE 15-HETE-d8 293 185 -70 -20
13-OxoODE 15-HETE-d8 293 113 -70 -20
9(10)-EpOME 15-HETE-d8 295 171.1 -70 -20
9-HODE 15-HETE-d8 295 171 -115 -23
13-HODE 15-HETE-d8 295 195 -115 -23
Table S4. LC-MS acquisition parameters for lipidomics assay in negative ionization mode.
Lipid INTERNAL STANDARD Q1 (m/z) Q3
(m/z)
CE (V)
PA(15:0/18:1(d7)) INTERNAL STANDARD 666.52 241.3 -50
PA(16:0_18:1) PA(15:0/18:1(d7)) 673.5 255.3 -50
PA(18:0_18:1) PA(15:0/18:1(d7)) 701.5 283.3 -50
PA(18:1/18:1) PA(15:0/18:1(d7)) 699.5 281.3 -50
PA(18:0_20:4) PA(15:0/18:1(d7)) 723.5 283.3 -50
PA(18:0_22:6) PA(15:0/18:1(d7)) 747.5 283.3 -50
PE(15:0/18:1(d7)) INTERNAL STANDARD 709.6 241.3 -50
PE(P-18:0/18:1) PE(15:0/18:1(d7)) 728.6 281.3 -50
PE(P-18:0_18:2) PE(15:0/18:1(d7)) 726.6 279.2 -50
PE(P-16:0_20:4) PE(15:0/18:1(d7)) 722.6 303.3 -50
PE(P-18:0_20:4) PE(15:0/18:1(d7)) 750.6 303.3 -50
PE(P-16:0_22:6) PE(15:0/18:1(d7)) 746.6 327.3 -50
PE(P-18:0_22:6) PE(15:0/18:1(d7)) 774.6 327.3 -50
(3-O-sulfo)Gal-Cer(d18:1/12:0) INTERNAL STANDARD 722.5 97 -150
(3-O-sulfo)Gal-Cer(d18:1/16:0) (3-O-sulfo)Gal-Cer(d18:1/12:0) 778.5 97 -150
(3-O-sulfo)Gal-Cer(d18:1/18:0) (3-O-sulfo)Gal-Cer(d18:1/12:0) 806.6 97 -150
(3-O-sulfo)Gal-
Cer(d18:1/18:0(2OH)
(3-O-sulfo)Gal-Cer(d18:1/12:0) 822.6 97 -150
(3-O-sulfo)Gal-Cer(d18:1/24:0) (3-O-sulfo)Gal-Cer(d18:1/12:0) 890.7 97 -150
(3-O-sulfo)Gal-
Cer(d18:1/24:0(2OH))
(3-O-sulfo)Gal-Cer(d18:1/12:0) 906.7 97 -150
(3-O-sulfo)Gal-Cer(d18:1/24:1) (3-O-sulfo)Gal-Cer(d18:1/12:0) 888.7 97 -150
(3-O-sulfo)Gal-
Cer(d18:1/24:1(2OH))
(3-O-sulfo)Gal-Cer(d18:1/12:0) 904.7 97 -150
LPE(18:1(d7)) INTERNAL STANDARD 485.3 288.3 -50
LPE(P-16:0) LPE(18:1(d7)) 436.3 196.1 -50
LPE(P-18:0) LPE(18:1(d7)) 464.3 196.1 -50
LPE(P-18:1) LPE(18:1(d7)) 462.3 196.1 -50
LPE(16:0) LPE(18:1(d7)) 452.2 255.3 -50
LPE(18:0) LPE(18:1(d7)) 480.31 283.3 -50
LPE(18:1) LPE(18:1(d7)) 478.3 281.3 -50
LPI(16:0) LPE(18:1(d7)) 571.3 241.1 -50
LPI(18:0) LPE(18:1(d7)) 599.3 241.1 -50
LPI(20:4) LPE(18:1(d7)) 619.3 241.1 -50
LPG(16:0) LPE(18:1(d7)) 483.3 255.3 -50
LPG(18:0) LPE(18:1(d7)) 511.3 283.3 -50
LPG(18:1) LPE(18:1(d7)) 509.3 281.3 -50
LPG(20:4) LPE(18:1(d7)) 531.3 303.3 -50
CL(14:0/14:0/14:0/14:0) INTERNAL STANDARD 619.5 227.2 -50
CL(72:8) CL(14:0/14:0/14:0/14:0) 723.7 279.3 -50
Cholesterol Sulfate 3-O-sulfo)Gal-Cer(d18:1/180(d3)) 465.3 96.7 -80
PG(15:0_18:1(d7)) INTERNAL STANDARD 740.55 241.3 -50
PG(16:0_18:1) PG(15:0_18:1(d7)) 747.5 255.3 -50
PG(18:0_18:1) PG(15:0_18:1(d7)) 775.5 283.3 -50
PG(18:1/18:1) PG(15:0_18:1(d7)) 773.5 281.3 -50
PG(18:0_20:4) PG(15:0_18:1(d7)) 797.5 283.3 -50
PI(15:0/18:1(d7)) INTERNAL STANDARD 828.6 241.3 -50
PI(18:0_18:1) PI(15:0/18:1(d7)) 863.6 283.3 -50
PI(16:0_20:4) PI(15:0/18:1(d7)) 857.6 255.3 -50
PI(18:0_20:4) PI(15:0/18:1(d7)) 885.6 283.3 -50
PI(16:0_22:6) PI(15:0/18:1(d7)) 881.6 255.3 -50
PI(18:0_22:6) PI(15:0/18:1(d7)) 909.6 283.3 -50
PI(20:4/20:4) PI(15:0/18:1(d7)) 905.6 303.3 -50
PS(15:0/18:1(d7)) INTERNAL STANDARD 753.55 241.3 -50
PS(18:0_18:1) PS(15:0/18:1(d7)) 788.6 283.3 -50
PS(18:0_20:4) PS(15:0/18:1(d7)) 810.6 283.3 -50
PS(16:0_22:6) PS(15:0/18:1(d7)) 806.6 255.3 -50
PS(18:0_22:6) PS(15:0/18:1(d7)) 834.6 283.3 -50
PS(22:6/22:6) PS(15:0/18:1(d7)) 878.5 327.3 -50
Arachidonic acid(d8) INTERNAL STANDARD 311.3 311.3 -10
Palmitic acid Arachidonic acid(d8) 255.1 255.1 -10
Palmitoleic acid Arachidonic acid(d8) 253.1 253.1 -10
Stearic acid Arachidonic acid(d8) 283.2 283.2 -10
Oleic acid Arachidonic acid(d8) 281.2 281.2 -10
Linoleic acid Arachidonic acid(d8) 279.2 279.2 -10
Linolenic acid Arachidonic acid(d8) 277.2 277.2 -10
Arachidonic acid Arachidonic acid(d8) 303.2 303.2 -10
EPA Arachidonic acid(d8) 301.2 301.2 -10
DHA Arachidonic acid(d8) 327.2 327.2 -10
Table S5. LC-MS acquisition parameters for lipidomics assay in positive ionization mode.
Lipid INTERNAL STANDARD Q1 (m/z) Q3 (m/z) DP (V) CE
Sphingosine (d17:1) INTERNAL STANDARD 286.2 268.3 80 20
Sphingosine Sphingosine(d17:1) 300.2 282.2 80 20
Sphinganine Sphingosine(d17:1) 302.2 284.2 80 20
Glucosyl sphingosine(d5) INTERNAL STANDARD 467.2 269.3 45 16
Hexosyl sphingosine Glucosyl sphingosine(d5) 462.3 282.2 45 16
Lactosyl sphingosine Glucosyl sphingosine(d5) 624.4 282.3 45 16
Cer(d18:1/17:0) INTERNAL STANDARD 552.4 264.3 80 40
Cer(d18:1/16:0) Cer(d18:1/16:0(d7)) 538.5 264.4 80 40
Cer(d18:1/18:0) Cer(d18:1/16:0(d7)) 566.6 264.4 80 40
Cer(d18:1/24:0) Cer(d18:1/16:0(d7)) 650.6 264.4 80 40
Cer(d18:1/24:1) Cer(d18:1/16:0(d7)) 648.6 264.4 80 40
SM(d18:1(d9)/18:1) INTERNAL STANDARD 738.7 184.1 80 40
SM(d18:1/16:0) SM(d18:1(d9)/18:1) 703.6 184.1 80 40
SM(d18:1/18:0) SM(d18:1(d9)/18:1) 731.6 184.1 80 40
SM(d18:1/24:0) SM(d18:1(d9)/18:1) 815.7 184.1 80 40
SM(d18:1/24:1) SM(d18:1(d9)/18:1) 813.7 184.1 80 40
LacCer(d18:1/16:0) GlcCer(d18:1(d5)/18:0) 862.6 264.6 80 40
LacCer(d18:1/18:0) GlcCer(d18:1(d5)/18:0) 890.7 264.4 80 40
LacCer(d18:1/24:0) GlcCer(d18:1(d5)/18:0) 974.8 264.4 80 40
LacCer(d18:1/24:1) GlcCer(d18:1(d5)/18:0) 972.7 264.4 80 40
LPC(18:1(d7)) INTERNAL STANDARD 529.3 184.1 80 40
LPC(16:1) LPC(18:1(d7)) 494.5 184.1 80 40
LPC(16:0) LPC(18:1(d7)) 496.3 184.1 80 40
LPC(18:0) LPC(18:1(d7)) 524.3 184.1 80 40
LPC(18:1) LPC(18:1(d7)) 522.3 184.1 80 40
LPC(20:4) LPC(18:1(d7)) 544.3 184.1 80 40
LPC(22:6) LPC(18:1(d7)) 568.3 184.1 80 40
LPC(24:1) LPC(18:1(d7)) 606.5 184.1 80 40
LPC(24:0) LPC(18:1(d7)) 608.5 184.1 80 40
LPC(26:1) LPC(18:1(d7)) 634.5 104.1 80 40
LPC(26:0) LPC(18:1(d7)) 636.5 104.1 80 40
Sphingosine-1-phosphocholine LPC(18:1(d7)) 465.5 184.1 80 40
Cholesterol(d7) INTERNAL STANDARD 376.2 376.2 80 10
Cholesterol Cholesterol(d7) 369.3 369.3 80 10
CE(18:1(d7)) INTERNAL STANDARD 675.2 369.4 80 26
CE(16:1) CE(18:1(d7)) 640.6 369.3 80 26
CE(18:1) CE(18:1(d7)) 668.6 369.3 80 26
CE(18:2) CE(18:1(d7)) 666.6 369.3 80 26
CE(20:4) CE(18:1(d7)) 690.6 369.3 80 26
CE(20:5) CE(18:1(d7)) 688.6 369.3 80 26
CE(22:6) CE(18:1(d7)) 714.6 369.3 80 26
TG(15:0/18:1(d7)/15:0) INTERNAL STANDARD 829.4 523.5 80 40
TG(52:4/18:1) TG(15:0/18:1(d7)/15:0) 872.7 573.4 80 40
TG(52:3/18:1) TG(15:0/18:1(d7)/15:0) 874.7 575.4 80 40
TG(54:2/18:0) TG(15:0/18:1(d7)/15:0) 904.7 603.4 80 40
TG(52:5/20:4) TG(15:0/18:1(d7)/15:0) 870.6 549.3 80 40
TG(54:6/20:4) TG(15:0/18:1(d7)/15:0) 896.6 575.3 80 40
TG(54:7/20:4) TG(15:0/18:1(d7)/15:0) 894.6 573.3 80 40
TG(56:4/20:4) TG(15:0/18:1(d7)/15:0) 928.8 607.5 80 40
TG(56:6/20:4) TG(15:0/18:1(d7)/15:0) 924.7 603.4 80 40
TG(56:7/20:4) TG(15:0/18:1(d7)/15:0) 922.7 601.4 80 40
TG(58:5/20:4) TG(15:0/18:1(d7)/15:0) 954.7 633.4 80 40
TG(58:7/20:4) TG(15:0/18:1(d7)/15:0) 950.7 629.4 80 40
TG(58:8/22:6) TG(15:0/18:1(d7)/15:0) 948.7 603.4 80 40
TG(60:7/22:6) TG(15:0/18:1(d7)/15:0) 978.7 633.4 80 40
TG(60:8/22:6) TG(15:0/18:1(d7)/15:0) 976.7 631.4 80 40
Sphingosine-1-phosphate(d17:1) INTERNAL STANDARD 366.3 250.3 80 25
Sphingosine-1-phosphate Sphingosine(d17:1) 380.3 264.3 80 25
GB3(d18:1/16:0) GlcCer(d18:1(d5)/18:0) 1025 520.5 80 40
GB3(d18:1/18:0) GlcCer(d18:1(d5)/18:0) 1053 548.6 80 40
GB3(d18:1/24:0) GlcCer(d18:1(d5)/18:0) 1137 632.6 80 40
GB3(d18:1/24:1) GlcCer(d18:1(d5)/18:0) 1135 630.6 80 40
Glc-Cholesterol CE(18:1(d7)) 566.6 369.3 80 17
Glc-Sitosterol CE(18:1(d7)) 594.6 397.4 80 17
Hydroxy cholesterol CE(18:1(d7)) 385.3 367.3 80 30
DG(16:0_18:1) DG(15:0/18:1(d7)) 614.4 313.4 80 30
DG(18:0_18:1) DG(15:0/18:1(d7)) 640.4 341.3 80 30
DG(18:1_8:1) DG(15:0/18:1(d7)) 638.4 339.3 80 30
DG(16:0_20:4) DG(15:0/18:1(d7)) 634.5 313.3 80 30
DG(18:0_20:4) DG(15:0/18:1(d7)) 662.5 341.3 80 30
DG(18:0_22:6) DG(15:0/18:1(d7)) 686.6 341.3 80 30
DG(18:1_20:4) DG(15:0/18:1(d7)) 660.5 339.3 80 30
DG(15:0_18:1(d7)) INTERNAL STANDARD 605.6 346.5 80 30
MG(18:1(d7)) INTERNAL STANDARD 381.3 272.5 80 22
MG(16:0) MG(18:1(d7)) 348.3 239.3 80 22
MG(20:4) MG(18:1(d7)) 396.3 287.3 80 22
MG(18:0) MG(18:1(d7)) 376.3 267.3 80 22
MG(18:1) MG(18:1(d7)) 374.3 265.3 80 22
MG(22:6) MG(18:1(d7)) 403.3 311.3 80 22
PC(15:0/18:1(d7)) INTERNAL STANDARD 754.6 184.1 80 40
PC(36:1) PC(15:
0/18:1(d7))
788.6 184.1 80 40
PC(36:2) PC(15:0/18:1(d7)) 786.6 184.1 80 40
PC(36:4) PC(15:0/18:1(d7)) 782.6 184.1 80 40
PC(38:4) PC(15:0/18:1(d7))
810.6 184.1 80 40
PC(38:6) PC(15:0/18:1(d7)) 806.6 184.1 80 40
PC(40:4) PC(15:0/18:1(d7)) 838.6 184.1 80 40
PC(40:6) PC(15:0/18:1(d7)) 834.6 184.1 80 40
PC(O-18:0/2:0) LPC(18:1(d7)) 524.3 184.1 80 40
PE(15:0/18:1(d7)) INTERNAL STANDARD 711.6 570.5 80 40
PE(36:1) PE(15:0/18:1(d7)) 746.6 605.5 80 40
PE(36:2) PE(15:0/18:1(d7)) 744.6 603.5 80 40
PE(36:4) PE(15:0/18:1(d7)) 740.6 599.5 80 40
PE(38:4) PE(15:0/18:1(d7)) 768.6 627.5 80 40
PE(38:6) PE(15:0/18:1(d7)) 764.6 623.5 80 40
PE(40:4) PE(15:0/18:1(d7)) 796.6 655.5 80 40
PE(40:6) PE(15:0/18:1(d7)) 792.6 651.5 80 40
POV-PC PC(15:0/18:1(d7)) 594.5 184.1 80 40
PC(16:0/9:0(CHO)) PC(15:0/18:1(d7)) 650.4 184.1 80 40
PC(16:0/9:0(COOH)) PC(15:0/18:1(d7)) 666.4 184.1 80 40
LysoPAF(16:0) PC(15:0/18:1(d7)) 482.3 184.1 80 40
PC(O-16:0/2:0) PC(15:0/18:1(d7)) 524.3 184.1 80 40
PC(18:0/20:4(OH[S])) PC(15:0/18:1(d7)) 826.6 184.1 80 40
PC(18:0/20:4(OOH[S])) PC(15:0/18:1(d7)) 842.6 184.1 80 40
PE(18:0/20:4(OH[S])) PE(15:0/18:1(d7)) 784.5 643.4 80 40
PE(18:0/20:4(OOH[S])) PE(15:0/18:1(d7)) 800.5 659.4 80 40
Coenzyme Q10 TG(15:0/18:1(d7)/15:0) 863.3 197.2 100 35
Table S6. Targeted lipidomic analysis of IDS KO; TfRmu/hu KI mice after repeated
administration of ETV:IDS or idursulfase.
Analyte
IDS
KO;TfRmu/hu
Vehicle
IDS
KO;TfRmu/hu
Idursulfase
IDS
KO;TfRmu/hu
ETV:IDS
PA(16:0_18:1) 1.02 1.03 0.88
PA(18:0_18:1) 0.98 1.02 0.89
PA(18:1_18:1) 1.02 1.02 0.88
PA(18:0_20:4) 1.06 1.03 0.95
PA(18:1_22:6) 1.03 1.01 0.98
PA(18:0_22:6) 0.99 0.93 0.93
PE(P-18:0/18:1) 0.98 1.03 0.95
PE(P-18:0/18:2) 0.97 0.94 0.89
PE(P-16:0/20:4) 0.99 0.93 0.94
PE(P-18:0/20:4) 0.97 0.92 0.89
PE(P-16:0/22:6) 0.92 0.87 0.86
PE(P-18:0/22:6) 1.00 0.96 0.95
(3-O-sulfo)Ga1-Cer(d18:1/16:0) 0.91 0.76 0.79
(3-O-sulfo)Ga1-Cer(d18:1/18:0) 0.81 0.68 0.76
(3-O-sulfo)Ga1-Cer(d18:1/18:0(2OH) 0.76 0.65 0.76
(3-O-sulfo)Ga1-Cer(d18:1/24:0) 0.76 0.66 0.71
(3-O-sulfo)Ga1-Cer(d18:1/24:0(2OH)) 0.79 0.75 0.73
(3-O-sulfo)Ga 1-Cer(d18:1/24:1) 0.82 0.75 0.75
(3-O-sulfo)Ga 1-Cer(d18:1/24:1(2OH)) 0.67 0.57 0.63
GM3(d34:1) 2.09 1.98 1.17
GM3(d36:1) 3.74 2.98 1.08
GM3(d38:1) 5.52 4.12 1.00
GM3(d40:1) 1.42 1.40 1.03
GD3(d34:1) 1.47 1.31 0.96
GD3(d36:1) 1.47 1.22 0.86
GD3(d38:1) 1.54 1.21 0.82
GD3(d40:1) 0.50 0.77 0.56
GD3(d42:2) 1.24 1.22 1.00
GD3(d42:1) 1.37 1.31 1.06
GD1a/b(d36:1) 0.88 0.82 0.82
GD1a/b(d38:1) 0.94 0.86 0.82
GTlb(d36:1) 0.93 0.86 0.81
GTlb(d38:1) 1.01 0.99 1.06
GQ1b(d36:1) 5.41 4.17 1.00
GQ1b(d38:1) 0.91 0.91 1.05
BMP(20:4/20:4) 0.92 0.88 0.75
BMP(22:6/22:6) 1.36 1.32 0.97
BMP(18:1/18:1) 1.70 1.60 0.93
Palmitic acid 0.99 0.91 0.98
Palmitoleic acid 1.03 0.90 1.08
Stearic acid 0.96 0.86 0.92
Oleic acid 1.04 0.94 1.08
Linoleic acid 1.17 1.06 1.23
Linolenic acid 1.10 1.01 1.15
Arachidonic acid 0.96 0.89 0.93
EPA 1.16 1.11 1.09
DHA 1.13 1.06 1.14
LPE( P-16:0) 1.31 1.38 1.23
LPE(P-18:0) 1.30 1.29 1.17
LPE( P-18:1) 1.25 1.28 1.08
LPE(16:0) 1.18 1.19 1.02
LPE(18:0) 1.21 1.20 1.07
LPE(18:1) 1.26 1.35 1.16
LP1(16:0) 1.18 1.20 1.01
LPl(18:0) 1.23 1.22 1.00
LPl(20:4) 1.09 1.15 1.07
LPG(16:0) 1.21 1.22 1.04
LPG(18:0) 1.21 1.16 1.00
LPG(18:1) 1.20 1.22 1.04
LPG(20:4) 1.20 1.17 0.98
CL(72:8) 1.31 1.50 1.17
Cholesterol Sulfate 0.81 0.91 0.94
PG(16:0_18:1) 0.93 0.91 0.83
PG(18:0_18:1) 1.03 1.00 0.96
PG(18:1/18:1) 0.96 0.95 0.90
PG(18:0_20:4) 0.91 0.88 0.83
Pl(18:0_18:1) 1.18 1.13 1.06
Pl(18:1/18:1) 1.14 1.11 1.01
Pl(16:0_20:4) 1.21 1.15 1.11
Pl(18:0_20:4) 1.18 1.14 1.09
Pl(16:0_ 22:6) 1.18 1.15 1.01
Pl(18:0_22:6) 1.31 1.28 1.12
Pl(20:4/20:4) 1.32 1.46 1.26
PS(18:0_18:1) 1.04 0.95 0.91
PS(18:0_20:4) 1.07 1.00 0.94
PS(16:0_22:6) 1.01 0.92 0.88
PS(18:1_22:6) 1.03 0.95 0.93
PS(18:0_ 22:6) 0.92 0.88 0.84
Sphingosine 1.21 1.07 1.03
Sphinganine 1.13 1.01 0.96
Lactosylsphingosine 1.24 1.40 1.77
Cer(d18:1/16:0) 1.19 1.10 1.27
Cer(d18:1/18:0) 1.06 0.94 1.05
Cer(d18:1/24:0) 1.08 0.92 0.98
Cer(d18:1/24 :1) 0.83 0.88 0.81
SM(d18:1/16:0) 1.13 1.08 1.04
SM(d18:1/18:0) 0.92 0.83 0.86
SM(d18:1/24:0) 1.06 1.00 0.88
SM(d18:1/24:1) 1.12 1.04 1.03
LacCer(d18:1/16:0) 0.90 0.84 0.87
LacCer(d18:1/18:0) 0.86 0.81 0.78
LacCer(d18:1/24:0) 0.87 0.78 0.80
LacCer(d18:1/24:1) 1.16 0.98 0.90
LPC(16:0) 1.11 1.24 1.12
LPC(18:0) 1.11 1.16 1.08
LPC(18:1) 1.12 1.24 1.05
LPC(20:4) 0.98 1.13 0.96
LPC(22:6) 0.97 1.06 0.87
Lyso-Sphingomyelin (d18:1) 0.91 1.03 0.76
PC(36:1) 1.00 0.94 0.93
PC(36:2) 0.99 0.96 0.94
PC(36 :4) 0.96 0.92 0.92
PC(38:4) 0.96 0.92 0.93
PC(38:6) 0.96 0.92 0.91
PC(40:4) 0.92 0.86 0.91
PC(40:6) 0.95 0.91 0.90
PC(0-16:0/2:0) 1.15 1.20 1.02
PE(36:1) 1.06 1.05 1.02
PE(36:2) 1.15 1.13 1.05
PE(36:4) 1.05 1.07 1.04
PE(38:4) 1.04 1.02 0.99
PE(38:6) 1.07 1.06 0.99
PE(40:4) 1.05 1.02 1.05
PE(40:6) 1.04 1.04 0.99
Cholesterol 0.92 0.91 0.83
CE(16:1) 0.80 0.94 1.03
CE(18:1) 0.72 0.77 1.06
CE(18:2) 0.87 0.80 0.91
CE(20:4) 1. 42 1.39 1.51
CE(20:5) 0.48 0.53 0.98
CE(22:6) 0.47 0.46 1.14
TG(52:4/18:1) 0.94 0.66 3.79
TG(52:3/18:1) 1.09 0.85 3.47
TG(54:2/18:0) 1.08 1.02 1.06
TG(52:5/20:4) 0.95 0.95 0.94
TG(54:6/20:4) 0.95 0.88 1.37
TG(54:7/20:4) 0.97 0.86 1.54
TG(56:4/20:4) 0.74 0.66 0.61
TG (56:6/20:4) 0.96 0.99 0.87
TG (56:7/20:4) 0.92 0.88 1.13
TG(58:5/20:4) 0.93 0.95 0.82
TG(58:7/20:4) 0.94 0.89 0.85
TG(58:8/22:6) 1.08 1.00 0.94
TG (60:7/22:6) 0.97 0.82 0.73
TG(60:8/22:6) 1.05 0.99 0.92
Sphingosine-1-phosphate 0.82 0.75 0.73
GB3(d18:1/16:0) 2.71 1.24 2.32
GB3(d18:1/18:0) 1.73 1.42 0.82
GB3(d18:1/24:0) 1.25 0.98 0.90
GB3(d18:1/24:1) 1.24 1.70 1.99
LPC(26:0) 0.88 0.92 0.67
LPC(24:0) 1.09 1.08 0.86
LPC(26:1) 1.21 1.16 0.89
LPC(24:1) 1.08 1.10 0.83
LPC(16:1) 1.07 1.19 1.01
DCER(d18:0/16:0) 1.40 1.14 1.30
DCER(d18:0/18:0) 0.98 0.94 0.93
DCER(d18:0/24:0) 0.99 0.97 0.99
DCER(d18:0/24:1) 1.11 1.07 1.04
Glc-Cholesterol 1.17 1.29 1.06
Glc-Sitosterol 0.94 1.06 0.96
MG(18:1) 1.23 1.08 1.32
MG(20:4) 1.37 1.17 0.96
MG(18:0) 0.93 0.91 0.93
MG(16:0) 1.00 0.96 1.03
MG(22:6) 1.34 1.16 1.06
DG(18:0_18:1) 1.02 1.02 0.92
DG(18:1/18:1) 1.07 1.16 0.95
DG(16:0_20:4) 1.06 1.17 0.83
DG(18:0_20:4) 1.07 1.17 0.85
DG(18:0_22:6) 1.15 1.32 0.95
DG(18:1_20:4) 1.04 1.20 0.87
GlcCer(d18:1/16:0) 2.22 1.78 1.19
GlcCer(d18:1/18:0) 1.46 1.36 0.96
GlcCer(d18:2/18:0) 1.57 1.32 0.92
GlcCer(d18:1/20:0) 1.60 1.43 1.10
GlcCer(d18:2/20:0) 1.71 1.40 1.08
GlcCer(d18:1/22:0) 2.03 1.75 1.14
GlcCer(d18:1/22:1) 1.18 1.10 1.09
GlcCer(d18:2/22:0) 1.10 1.31 1.02
GlcCer(d18:1/24:1) 1.64 1.42 1.06
GlcCer(d18:1/24:0) 1.91 1.63 1.10
Ga ICer(d18:1/16:0) 0.95 0.99 1.04
Ga I Cer(d18:1/18:0) 1.08 1.08 1.07
Ga ICer(d18:2/18:0) 1.11 1.03 1.07
Ga ICer(d18:1/20:0) 0.98 1.00 1.01
Ga ICer(d18:2/20:0) 0.90 0.86 0.91
Ga ICer(d18:1/22:0) 0.95 0.98 0.96
GalCer(d18:1/22:1) 0.88 0.91 0.95
Ga ICer(d18:2/22:0) 0.87 0.84 0.84
GalCer(d18:1/24:1) 0.95 0.93 0.91
Ga ICer(d18:1/24:0) 0.96 0.94 0.91
alpha-GalCer(d18:1/16:0) 0.89 0.75 0.80
Glucosylsphingosine 1.11 0.71 0.94
Galactosylsphingosine 0.99 0.85 0.97
5-HETE 1.05 0.97 1.25
12-HETE 1.27 0.91 1.40
15-HETE 1.39 1.18 1.57
6k-PGF1alpha 1.12 1.01 1.27
TxB2 1.66 1.45 1.72
PGF2alpha 1.13 1.05 1.36
PGE2 1.58 1.36 1.52
5,15-DiHETE 1.48 1.13 1.40
5-iso-PGF2alpha 1.45 1.42 1.37
PGD2 1.79 1.48 1.34
9-HOTrE 1.18 1.14 1.09
9-OxoODE 1.02 1.00 1.29
13-OxoODE 0.85 0.84 0.87
9(10)-EpOME 0.99 1.01 0.95
9-HODE 1.36 1.17 1.52
13-HODE 1.14 0.96 1.27
IDS KO; TfRmu/hu KI mice were administered four weekly intravenous injections of 40 mg/kg
ETV:IDS or 14.2 mg/kg idursulfase (747 µmol product/min/kg activity equivalent dose) as
described. Levels of analytes from a targeted lipid panel were measured from the brains of wild-
type mice, IDS KO; TfRmu/hu KI mice treated with vehicle, ETV:IDS or idursulfase. Analytes
were analyzed using LC-MS/MS, and levels were calculated by normalizing to known amounts
of internal standards and total brain weight before homogenization (resulting in ng/mg lipid).
Table shows the group average and SEM; n=8 per IDS KO; TfRmu/hu KI group and n=5 for wild-
type group) of each lipid species, including the sum (SUM) of each class of lipids.
Data file S1. Raw data.
Provided as a separate Excel file.