Akiyama 2014. Enzyme Augmentation Therapy Enhances the Therapeutic Efficacy of Bone Marrow Transplantation in Mucopolysaccharidosis Type II Mice

8/11/2019 Akiyama 2014. Enzyme Augmentation Therapy Enhances the Therapeutic Efficacy of Bone Marrow Transplantatio

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Enzyme augmentation therapy enhances the therapeutic efcacy of bone marrow

transplantation in mucopolysaccharidosis type II mice

Kazumasa Akiyama a,b, Yohta Shimada a,Takashi Higuchi a, Makoto Ohtsu c, Hiromitsu Nakauchi c,Hiroshi Kobayashi a,d, TakahiroFukuda e, Hiroyuki Ida a,d, Yoshikatsu Eto f, Brett E. Crawford g,Jillian R. Brown g, Toya Ohashi a,d,a Department of Gene Therapy, Institute of DNA Medicine, Research Center for Medical Sciences, The Jikei University School of Medicine, Tokyo, Japanb Department of Pediatrics, Kitasato University Graduate School of Medicine, Kanagawa, Japanc Division of Stem Cell Therapy, Center for Stem Cell and Regenerative Medicine, Institute of Medical Science, University of Tokyo, Tokyo, Japand Department of Pediatrics, The Jikei University School of Medicine, Tokyo, Japane Division of Neuropathology, Department of Pathology, The Jikei University School of Medicine, Tokyo, JapanfAdvanced Clinical Research Center, Institute of Neurological Disorders, Kanagawa, Japang BioMarin Pharmaceutical Inc., San Diego, CA, USA

a b s t r a c ta r t i c l e i n f o

Article history:

Received 5 August 2013

Received in revised form 17 September 2013

Accepted 17 September 2013

Available online 21 September 2013

Keywords:

Mucopolysaccharidosis

Bone marrow transplantation

Enzyme replacement therapy

GlycosaminoglycanPathologic glycosaminoglycan

Hunter syndrome

Before the availability of an enzyme replacement therapy (ERT) for mucopolysaccharidosis type II (MPS II),

patients were treated by bone marrow transplantation (BMT). However, the effectiveness of BMT for MPS II

was equivocal, particularly at addressing the CNS manifestations. To study this further, we subjected a murine

model of MPS II to BMT and evaluated the effect at correcting the biochemical and pathological aberrations in

the viscera and CNS. Our results indicated that BMT reduced the accumulation of glycosaminoglycans (GAGs)

in a variety of visceral organs, but not in the CNS. With the availability of an approved ERT for MPS II, we inves-

tigated and compared the relative merits of the two strategies either as a mono or combination therapy.

We showed that the combination of BMT and ERT was additive at reducing tissue levels of GAGs in the heart,

kidney and lung. Moreover, ERT conferred greater efcacy if the immunological response against the infused

recombinant enzyme was low. Finally, we showed that pathologic GAGs might potentially represent a sensitivebiomarker to monitor the therapeutic efcacy of therapies for MPS II.

2013 Elsevier Inc. All rights reserved.

1. Introduction

Mucopolysaccharidosis type II (MPS II, Hunter syndrome) is an

X-linked lysosomal storage disorder (LSD) caused by a deciency in

the activity of the lysosomal enzyme, iduronate-2-sulfate (IDS, EC

3.1.6.13), which degrades the glycosaminoglycans (GAGs), heparan

sulfate and dermatan sulfate[1]. The widespread and progressive lyso-

somal accumulation of undegraded GAGs leads to a broad spectrum

of clinical manifestations. These include skeletal deformities, cardiac

valvular disease, cardiac hypertrophy, hepatosplenomegaly, coarse fa-

cial appearance, upper airway narrowing, hearing defect, enlarged

tongue, retinopathy and CNS involvement[2]. These clinical symptoms

signicantly compromise the patients' quality of life.

Presently, two therapies are available to treat MPS II; one is enzyme

replacement therapy (ERT) and the other is bone marrow transplanta-

tion (BMT). ERT has been shown to be effective at correcting aspects

of the visceral disease[35]but not the CNS lesion as the enzyme dose

not cross the bloodbrain barrier[6,7]. ERT also has a limited impact

on the bone and valvular lesions [8].In addition, the need for repeated

infusions of the enzyme is costly and confers a heavy burden on the

MPS II patients[9].

BMT has been shown to be effective at treating several neuropathic

LSDs such as MPS I, MPS VI, globoid-cell leukodystrophy, metachro-

matic leukodystrophy, Gaucher disease and others[10,11]. In contrast

to ERT, BMT might address the CNS lesions associated with several

LSDs by virtue of the migration of enzyme competent donor cells into

the brain. Consequent secretion of the enzyme from these cells may

allow for cross-correction of patients' enzyme decient neuronal cells.

Early studies suggested that BMT should not be indicated for MPS II

patients due to disappointing outcomes, particularly the limited impact

on CNS involvement [1215]. However, recent report demonstrated the

long-term efcacy of BMT in CNS involvement of an MPS II patient [16].

Thus, it is not evident if BMT should be indicated for MPS II. Moreover,

there have been no animal studies showing the impact of BMT at reduc-

ing the level of GAGs in the brain.

Prior to the availability of ERT, some patients were treated by BMT

with the hope of improving the visceral disease and CNS disease. Now

Molecular Genetics and Metabolism 111 (2014) 139146

Corresponding author at: 3-25-8 Nishishinbashi, Minato-ku, Tokyo 105-8461, Japan.

Fax: +81 3 3433 1230.

E-mail address:[email protected](T. Ohashi).

1096-7192/$ see front matter 2013 Elsevier Inc. All rights reserved.

http://dx.doi.org/10.1016/j.ymgme.2013.09.013

Contents lists available atScienceDirect

Molecular Genetics and Metabolism

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that an approved enzyme is available for these patients, we asked if

ERT could enhance the therapeutic effect of BMT. This has not been

demonstrated for either human or murine MPS II. In this study, we

examined the effectiveness of BMT at addressing CNS disease, as well

as the relative merits of the combination of BMT and ERT in a mouse

model of MPS II.

2. Methods

2.1. Animal husbandry

Female mice heterozygous for the X-linked allele (IDS+/) on a

congenic C57BL/6 background were kindly provided from Joseph

Muenzer (University of North Carolina, Chapel Hill) [17].The carrier fe-

males were bred with male wild type (WT) mice of the same genetic

background strain, producing hemizygous IDS knock-out males (MPS II

mouse model, IdS/0). The genotypes of all offspring were determined

by polymerase chain reaction analysis of DNA obtained from a tail

snip. B6.SJL-ptprca mice congenic at the CD45 locus (CD45.1+CD45.2)

were purchased from Sankyo Labo Service (Tokyo, Japan), and bred

with female C57BL/6 (CD45.1CD45.2+) WT, producing C57BL/6

(CD45.1+CD45.2+) donor mice. These mice were used as donors.

Animal husbandry and all procedures in the animal experiments wereapproved by The Animal Care Committee at The Jikei University School

of Medicine.

2.2. BMT

BMT was performed as previously described with minor modica-

tion[18]. Bone marrow cells were harvested from femurs and tibias

of male C57BL/6 (CD45.1+CD45.2+) mice (812 weeks of age). The

bone marrow cells (2.0 106) were transplanted to lethally irradiated

(9Gy) recipient MPS II mice intravenously. Irradiation was carried

out using the Hitach-MBR1520R irradiator (Hitachi, Tokyo, Japan). To

conrm engraftment, the peripheral blood was collected from treated

mice (2 times: 12 weeks and 27 weeks after transplantation). Flow

cytometric analysis was carried out using MACSQuant Analyzer(Miltenyi Biotec, Bergisch Gladbach, Germany) and analyzed using

MACSQuantify Software (Miltenyi Biotec). Briey, peripheral blood

cells were stained with uorescein isothiocyanate-conjugated anti-

murine CD45.1 and allophycocyanin-conjugated anti-murine CD45.2

(eBioscience, San Diego, CA, USA). The each lineage was distinguished

by using the corresponding phycoerythrin-conjugated antibody: (Bcell-

CD45R, Tcell-CD3e, Granulocyte-Ly6G, Macrophage-CD11b, eBioscience).

The donor-derived cell engraftment was determined as the percentage

of CD45.1+CD45.2+ cells.

2.3. ERT

The MPS II mice were administered 0.5 mg/kg human IDS

(Idursulfase, Shire HGT Pharmaceuticals, Cambridge, MA, USA, gener-

ously gifted from Genzyme Japan Co., Ltd. in Tokyo) via a tail vein

once a week.

2.4. Therapeutic regimen

Therapeutic regimen was shown in Fig. 1. There are 3 treatment

groups, BMT group, ERT group and BMT + ERT group. Control groups

consisted with untreated MPS II mice and WT mice. In ERT group, ERT

started at 9 weeks of age with total 27 times. In BMT group, BMT was

carried out at 9 weeks of age.In BMT + ERTgroup, BMT was performed

at 9 weeks of age and ERT was initiated at 12 weeks after BMT with

total 15 times. All mice were measured the body weight and collected

the blood and urine samples at various time points. In ERT and

BMT + ERT groups, blood was harvested just prior to ERT every time.

Y-maze test was carried out in all treatment groups at between 26

and 27 weeks after treatment, and sacriced for tissue biochemical

and pathological assays at 27 weeks after initiation of treatment. The

mice in ERT and BMT + ERT groups sacriced 1 week after last ERT.

2.5. Serum and tissue IDS activity

IDS activity was determined in serum and homogenized tissue, as

previously described using the articial substrate 4-methylumbelliferyl-

alpha-iduronide-2-sulfate (MU-Idu-2S) (Moscerdam Substrates,

Oegstgeest, Netherlands) [19]. Proteinconcentrations were determined

using the BCA protein assay kit (Thermo Fisher Scientic, IL, USA). The

tissue IDS enzyme activity was expressed as nmol/4 h/mg protein.

The serum IDS activity was expressed as % activity of WT mice.

2.6. Total GAGs assay

Total GAG (tGAG) concentration in urine and tissue extracts was

quantied by the Wieslad sGAG quantitative Alcian blue-binding

assay kit (Euro-Diagnostica, Malm, Sweden) as previously described

[19].The urine creatinine was assayed using a commercially available

kit (Wako Pure Chemical Industries, Ltd., Osaka, Japan). The urinary

and tissue GAG amount was expressed as g GAG/mg creatinine and

g GAG/mg protein, respectively.

Fig. 1. Therapy experimentaldesign. Thepictureshowsdiagram oftherapeuticregimen. Thetherapeuticgroups included BMTonly, ERTonly, andthe combinationof BMTand ERTin MPS

II mice. Downward triangles mark the initiation of BMT. The solid arrows indicate IDS administration. Open circles mark time points of blood and urine collection for IDS activity, GAG

detection, and anti-IDS antibody assays. The lled circles show the time point of sacrice. In BMT groups, BMT was carried out at 9 weeks of age. In ERT group, ERT was initiated at

9 weeks of age followed by weekly infusion total 27 times. In ERT + BMT groups, BMT was carried out at 9 weeks of age and ERT was added 12 weeks after BMT. Thereafter, ERT was

continued weekly for a total of 15 times. All mice were sacriced 27 weeks after initiation of treatment.

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2.7. Pathologic GAGs assay

The pathologic GAGs (pGAGs) assays were performed by using

the Sensi-Pro Non-Reducing End (NRE) heparan sulfate assay as de-

scribed[20]. The Sensi-Pro assay measures only lysosomal accumu-

lated heparan sulfate caused by the decient activity of IDS by

specically quantifying the 2-O sulfated non-reducing end derived

after heparin lyase digestion. Briey, tissue samples were homoge-

nized and the GAGs were isolated by anion exchange chromatogra-phy and digested with heparin lyase (IBEX Technologies, Montreal,

Canada). Enzymatically depolymerized MPS II specic NRE, 2-sulfo-

iduronic linked to N-sulfoglucosamine-6-sulfate (I2S6), tagged with

[12C6] was measured according to standard curves of saturated heparan

sulfate tagged with [13C6], derived commercially available standard

unsaturated disaccharides. This measurement was performed by using

Liquid ChromatographyMass Spectrometry and the data were repre-

sented as average of triplicates.

2.8. Pathologically analysis

The mice were euthanized using pentobarbital and perfused with

4% paraformaldehyde and 2% glutaraldehyde (Wako Pure Chemical

Industries, Ltd.) in 0.1 M PBS through the heart. The liver, heart, spleen

and kidney were removed and immersed in the same xative solution

for a minimum of 2 h. Tissue samples were postxed in 1% osmium

tetroxide, and embedded in epoxy resin. The sections were cut at 1 m

thickness, stained with toluidine blue, and assessed to detect large

distended lysosomes using light microscopy (BX50; Olympus Optical

Co, Ltd., Tokyo, Japan).

2.9. Assay for rhIDS-specic IgG

IgG antibodies recognizing rhIDS were assayed using an enzyme-

linked immunosorbent assay (ELISA). Briey, 96-well plates Nunc-

Immuno palate MaxiSorp (Nunc, Roskilde, Denmark) were coated

with 10 g of rhIDS in PBS overnight at 4 C. The plates were blocked

by adding 100 l PBS/1% bovine serum albumin and incubating

for 5 h at room temperature. After this step, the wells were washedwith PBS/0.05% Tween 20. The serum samples from mice were diluted

100-fold with PBS/1% bovine serum albumin, and 100l diluted

serum was added to each well and incubated for 1 h at room tempera-

ture. The plate was then washed with thesame buffer and reacted with

100l of 5000-fold diluted peroxidase-conjugated anti-mouse IgG Ab

(Kirkegaard & Perry Labs, Gaithersburg, MA, USA). After incubation for

30 min at room temperature, the plates were washed again and color

was generated by the addition of 3,3,5,5-tetramethylbenzidine sub-

strate reagent (Kirkegaard & Perry Labs) for 10 min at room tempera-

ture. The reaction was stopped by adding 100 l of 0.6 N H2SO4, and

the optical density was measured at 450 nm using an ARVOMX/Light

plate reader (PerkinElmer, Waltham, MA, USA). The Ab titer was calcu-

lated using mouse anti-human IDS monoclonal antibody as a standard

(generously gifted by JCR pharmaceutical Co., Ltd.).

2.10. Statistical analysis

Data were assessed using GraphPad Prism software (GraphPad soft-

ware, Inc., La Jolla, CA, USA). T-test or one-way ANOVA with Tukey

Kramer's post testwas used. Signicance was considered to be Pb0.05.

3. Results

3.1. Engraftment of donor cells in mice treatedwith BMT and a combination

of BMT and ERT

Donor cell engraftment in mice subjected to BMT and BMT + ERT

was analyzed at 12 weeks and 27 weeks following BMT (Table 1).

Approximately 90% donor cell engraftment in each lineage cell was

achieved at 12 and 27 weeks in animals that received BMT alone or

in combination with ERT. Percent engraftment of T cell lineage was

lower than for other cell lineages probably because of the longer half

life time of pre-existing recipient T cells.

3.2. Serum IDS activity in mice treated with BMT, ERT or a combination of

BMT and ERT

Serum IDS activities at various time points were assayed following

treatment (Fig. 2). MPS II mice treated by BMT alone showed a rise in

IDS activity beginning at 2 weeks post-transplantation which reached

25% of WT levels at 20 weeks (P b0.001 vs. untreated MPS II mouse,

NT). Animals treated with ERT alone had no detectable serum IDS activ-

ity as the infused IDS was rapidly cleared from circulation. Serum IDS

levels in mice treated by the combination of BMT and ERT exhibited a

prole that was similar to mice treated by BMT alone (Pb0.001 vs.

NT, PN0.05 vs.BMT). As may be expected theserum IDS enzymatic ac-

tivity did not increase even after initiation of ERT as the infused enzyme

was rapidly cleared from circulation. These observations indicate that

the donor cells were stably engrafted in the BMT treated mice and

that they excreted IDS for a sustained period.

3.3. Tissue IDS activity in mice treated by BMT or ERT aloneand combination

of BMT and ERT

Twenty-sevenweeksafter treatment by BMTor ERT or thecombina-

tion, IDS activity in various tissues of MPS II mice was analyzed. In

the ERT treated group (Fig. 3A), no signicant increase in IDS activity

Table 1

Donor-derived cell engraftment in each lineage in BMT and BMT + ERT groups.

B cell (%)a T cell (%)a Granulocyte (%)a Macrophage (%)a

12 weeks after BMT

BMT 95.72 4.51 78.99 5.39 87.39 4.64 89.6 7.88

BMT + ERT 96.41 1.64 79.95 4.19 89.8 3.84 92.89 3.8

27 weeks after BMT

BMT 93.9 8.41 78.34 5.8 92.36 6.58 91.28 9.44

BMT + ERT 95.46 3.04 85.48 4.3 94.82 3.05 89.1 4.174a Percentage ofdonor-derivedcellsin peripheral bloodat 12or 27 weeksafterBMT.The

individual values are shown as mean values SD (BMT: n = 5, BMT + ERT: n = 5).

Fig. 2.Sustained IDS activities in serum from BMT received MPS II. IDS activity in serum

from MPS II mice in three treatment groups, WT mice and NT mice was measured as

described inMethods.The IDS activities were assayed duplicate. Arrows indicate start

point of ERT in BMT + ERT group. IDS activity is expressed as % of age matched WT

mice (data of WT mice is not shown in gure). The IDS activities at each time point

are shown as mean SEM (each group: n = 5). The difference of enzyme activities at

20 weeks after treatment was compared by one-way ANOVA among 3 treatment groups

and NT group. Asterisk indicatesPb

0.001.

n.s

indicates no signicant difference.

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was observed in any tissues tested, except the liver. As the tissues were

harvested one week after the last enzyme infusion, it is likely that the

enzyme had already turned over in most of tissues. In the liver, as

large amounts of the enzyme were taken up by the cells, we were still

able to detect some measure of activity even one week after the last

infusion. In the BMT-treated group (Fig. 3B), higher IDS activity was de-tected in the liver, heart, spleen, kidney and lung but not in cerebrum.

Micetreated by a combinationof BMT + ERT(Fig. 3C) showed a similar

prole of higher IDS activity. Unlike the BMT group, the increase of

IDS activity in the cerebrum was statistically signicant but the eleva-

tion of IDS activity was minimal. These results indicated that stable

engraftment of donor cells in recipient mice following BMT led to an

increase in IDS activity in various tissues.

3.4. Impact of elevated IDS activity on total tissue GAGs levels

To study the effect of BMT on the CNS disease, the potential for

an additive effect of a combination of ERT to BMT and to compare the

relative efcacy of BMT and ERT, we analyzed tGAGs levels in various

tissues. Analysis of GAGs using the Alcian blue method allows for a

determinationof tGAGs.In thecerebrum(Fig.4), none of the treatments

reduced tGAGs levels. However, because there was no signicant differ-

ence in cerebrum tGAGs levels between WT mice and NT MPS II mice,

this result is not informative. In liver (Fig. 4, Supplemental Fig. 1),

tGAGs were signicantly reduced by BMT, ERT and BMT + ERT com-

pared to untreated controls. However, there was no statistical differ-ence among the three treatment groups. There was a suggestion that

ERT provided an additive effect to BMT, but this trend was not statisti-

cally signicant. In the heart and spleen (Fig. 4, Supplemental Fig. 1),

all of the treatments reduced tGAGs to almost WT levels. There was

no statistical difference among three treatment groups and ERT did

not afford additive effect to BMT. In the kidney (Fig. 4, Supplemental

Fig. 1), ERT and BMT + ERT signicantly reduced tGAGs, but BMT

alone did not. ERT reduced tGAG levels more profoundly than BMT

and ERT conferred an additive effect to BMT. In the lung ( Fig. 4), all

treatment regimens reduced tGAGs signicantly. ERT reduced tGAGs

more profoundly than BMT and ERT conferred an additive effect to

BMT. Hence, an additive effect of ERT to BMT was observed in the

kidney and lung, and ERT was superior to BMT at reducing tGAGs in

the kidney and lung.

Fig. 3. Increasedtissue IDSenzymeactivity. Liver,heart,spleen, kidney, lung andcerebrum were harvestedfromtreatedmice,age matched WT mice andagematchedNT mice at27 weeks

afterinitiationof treatment. TissueIDS enzyme activity was measured as described in Methods. IDSactivityis expressedas nmol/4 h/mg protein.Data arepresentedas mean SEM(each

group: n = 5). Difference of enzyme activity between treatment group and NT group was compared using Student's t-test. Asterisk indicatesPb 0.05. n.s indicates no signicant

difference.

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3.5. Impact of elevated IDS activity on pathologic GAGs levels

Since there wasno additiveeffect ofERT to BMT atreducingtGAGs in

thecerebrum, liver,heart andspleen, we hadthe opportunity to utilize a

more sensitive and specic assay that detects only pathologic GAGs

(pGAGs, a selective measure of GAG fragments generated in the lyso-

some due to the deciency of IDS). In the cerebrum (Fig. 5), none of

treatments reduced pGAGs levels. It is worth noting though that the

difference in cerebrum pGAG levels between WT mice and NT MPS II

mice was more obvious than tGAGs. In the liver (Fig. 5), pGAGs were

signicantly reduced by BMT, ERT and BMT + ERT treatments com-

pared to untreated controls. However, the extent of reduction was not

statistically difference among the three treatment groups. Like noted

earlier with tGAGs, there was a suggestion that ERT conferred an addi-

tive effect to BMT; however the effect was not statistically different.

In the heart (Fig. 5), all the treatments signicantly reduced pGAGs.

ERT reduced pGAGs more profoundly than BMT and ERT provided an

additive effect to BMT.

In summary, none of the treatment reduced pGAGs in the cerebrum.

In the liver, the results with pGAG werethe sameas noted with tGAG. In

the heart, we demonstrated an additive effect of ERT to BMT but this

could not be proven in tGAGs. These results also indicated that pGAGs

are a better indicator than tGAGs of therapeutic effect of the treatments

in the cerebrum and heart.

3.6. Impact of elevated IDS activity on total urinary GAG levels

All the treatments reduced urinary tGAGs to almost normal levels

and no statistical difference among all treatment groups (Fig. 6). Thus,

Fig. 4. Corrections of total GAGs accumulation invarious tissues. The tGAGs in cerebrum, liver, heart, spleen, kidneyand lungwas assayed using Alcian blue method. Amount of tGAGs is

expressed as g/mg protein. Each symbol represents a data of individual mouse. Horizontal bar indicates mean value. Difference of tGAGs among 3 treatment groups and NT group was

compared using one-way ANOVA. Asterisk indicates P b0.05. n.sindicates no signicant difference.

Fig. 5. Pathologic GAGs accumulation in tissues. pGAGs were measured in cerebrum, liver and heart as described in Methods.The amount of pGAGs is expressed as pmols of MPS II

Sensi-Pro Marker per mg of tissue. Each symbol represents a data of individual mouse. Horizontal bar indicates mean value. Difference of pGAGs among 3 treatment groups and NT

group was compared using one-way ANOVA. Asterisk indicates Pb

0.05.

n.s

indicates no signicant difference.

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any additive effect of ERTto BMT or difference in efcacy between BMT

and ERT could not be observed.

3.7. Growth of MPS II mice

Body weights of mice were also monitored in each treatment

and control mice (Fig. 7). Similar to human MPS II patients[21], MPS

II mice also exhibited greater weights. All treatments suppressed

the overgrowth of MPS II mice when measured at 27 weeks post-

treatment. However, ERT did not provide an additive effect to BMT

and there was no statistically signicant difference between mice

treated by BMT and ERT.

3.8. IgG antibody against IDS in ERT groups

In some mice, anti IgG antibodies developed against the recombi-

nant enzyme (Supplemental Fig. 2). However, these antibody titers

were very low compared to titers developed following ERT in otherlysosomal storage disease mouse models. For example, when we

assayed the IgG antibody titer against infused enzyme in the mouse

model of Pompe disease employing a similar method to this study we

had to dilute serum 20,000 times or more to put in OD 450 nm values

in the linear range of standard curve [22]. However, in this study,

OD450 nm values were in linear range using 100 times diluted serum.

When the serum was diluted more, the antibody titer was under the

detection limit.

4. Discussion

In early 1980s, rst report described the effectiveness of BMT

for MPS I[23]. Subsequently, BMT was performed in many MPS I pa-

tients with encouraging results [24]. BMT was particularly effective

at addressing the CNS involvement of MPS I patients when BMT was

performed in patients who were b 2 years old and had an IQ70. Ani-

mal studies also demonstrated a reduction of GAGs in brain following

BMT[25]. In contrast to MPS I, there is huge controversy for indicatingBMT for MPS II patients [10,11]. BMT is not indicated for MPS II because

of the limited efcacy at correcting the CNS disease of MPS II patients

[1215]. However, a recent retrospective study of Japanese MPS II pa-

tients who received BMT demonstrated that this therapy was effective

at addressing the CNS disease of mild type MPS II (MPS IIB) and their

valvular disease [16]. However, asall the studies werein MPS II patients,

pathological and biochemical data are not available to support these

ndings. To address this issue, we performed BMT on MPS II mice to

clarify whether pathological, biochemical changes were evident. Our

studies indicated no evidence of an increase in enzyme activity or re-

duction of GAGs in the brain of transplanted MPS II mice. Pathological

analysis of brain sections was very hard to interpret because of the

limited amounts of storage material in the brain of MPS II mice (data

not shown). Moreover no neurofunctional improvement was observed

by BMT (data not shown). From these observations, we conclude

that BMT does not appear to be effective in treating the CNS disease

in MPS II, similar to MPS III[26]. However, it is possible that the assays

that we used here were not sufciently sensitive to monitor therapeu-

tic effects of the treatments to CNS involvement of MPS II. As an alter-

native approach to treat the CNS disease, other investigators are using

genetically-modied donor bone marrow cells to enhance the expres-

sion and secretion of the lysosomal enzyme prior to transplantation.

The effectiveness of this approach to treat the CNS disease has now

been reported in MPS III mice [27]. We did observe a reduction in

GAGs in many visceral diseases by BMT, as expected.

ERT for MPS II was approved in 2006 in US and EU, and in 2007

in Japan. ERT improved the performance of MPS II patients on the

6 minute walk test and their predicted FVC [35]. Although it is clear

that ERT offers a therapeutic effect, the cost of this therapy is veryhigh and requires lifelong repeated administration [9]. BMT has the pos-

sibility to overcome these problems. However, it is not known which

is more effective, BMT or ERT. From our study, ERT was more effective

than BMT at reducing GAGs in heart, kidney and lung. In studies of

ERT for LSDs other than MPS II, many reports suggested that antibodies

against the infused enzyme can inhibit the therapeutic efcacy of ERT,

and have been particularly well described in Pompe disease [28,29].

In our mice studies, antibody titers against the administered enzyme

were low; thus we might have overestimated the efcacy of ERT.

From our experience, if the immunological response against the infused

enzyme is controlled, ERT is superior to BMT, at least in a few organs.

Beforethe availability of ERT, BMTwas carried out in a small number

of MPS II patients. One report indicated that a single dose of ERT to al-

ready transplanted MPS VI patient caused further reduction of urinaryGAGs[30]. Another report indicated that supplementing BMT treated

MPSVI patients with ERTcausedfurther improvement of several clinical

parameters, including joint movement, outcome of the 12 minute walk

test and the outcome of the 3 minute stair-climbing test [31]. Several

studies reported that ERT in the peritransplanted period reduced the

BMT-related morbidity and mortality in children with MPS I and VI.

However, these studies addressed to safety issue of BMT not to additive

therapeutic effect of ERT to BMT[3236]. Moreover, MPS I and VI are

different diseases from MPS II. Thus, the results of MPS I and VI might

not relevant to MPS II. In this study, we introduced ERT to already

transplanted MPS II mice and found an additive effect of ERT to BMT

in the heart, kidney and lung, at least in terms of reducing GAG levels.

Taken together, an additive effect of ERT to BMT was observed in tissues

which ERT had a superior effect to BMT. From these observations, it

Fig. 6. Corrections of urinary total GAGs excretion. Urinary tGAGs were assayed using

Alcian blue method. Amount of urinary tGAGs is expressed as g/mg creatinine. The

data of each group are shown as mean SEM (WT: n = 7, BMT: n = 5, ERT: n = 5,

BMT + ERT: n = 5, NT: n = 6). Difference of tGAGs among 3 treatment groups and NT

group was compared using one-way ANOVA. Asterisk indicates Pb 0.05. n.sindicates

no signicant difference.

Fig. 7. Suppression ofovergrowthby treatment. Thebodyweightof mice wasmeasured at

every 3 weeks from 9 weeks of age until sacrice. The body weight of mice from each

group is shown as mean values. Difference of body weight at 27 weeks after treatment

among 3 treatment groups and NT group was compared using one-way ANOVA. n.s

indicates no signicant difference. (ERT or BMT or BMT + ERT vs NT; Pb 0.05, BMT vs

BMT + ERT; n.s, ERT vs BMT; n.s).

http://localhost/var/www/apps/conversion/tmp/scratch_9/image%20of%20Fig.%E0%B7%80http://localhost/var/www/apps/conversion/tmp/scratch_9/image%20of%20Fig.%E0%B6%80


7/8

might be worthwhile to perform ERT to already transplanted MPS II

patients, especially as the effects of BMT are limited. However, it is still

not known if more profound reduction of GAGs causes a more robust

improvement in clinical signs of MPS II. Hence, indicating ERT as a sup-

plement to BMT should be carefully determined.

Finally, we assayed tissue pGAGs and tGAGs to monitor the thera-

peutic effects of treatments in MPS II mice. From our observations,

there was very little difference between tGAGs in brain from WT and

MPS II mice (Fig. 4); however, there was a signi

cant difference be-tween pGAGs in brain from WT and MPS II mice (Fig. 5). Thus, like

MPS I[37], pGAGs are a better biomarker than tGAGs to monitor the

therapeutic effect in MPS II specically. These new pGAG quantitation

methods provide a simple, rapid diagnostic strategy for MPS I, II, IIIA,

IIIB, IIIC, IIID, VI and VII using samples of urine, blood and dried blood

spots. Analysis of the non-reducing end glycans provides a method for

monitoring not only enzyme replacement but also substrate reduction

therapies and serves as a discovery tool for uncovering novel bio-

markers and new forms of mucopolysaccharidoses[38]. In conclusion,

BMT and ERT alone or even as a combination, have a limited positive

effect on the brain disease of MPS II mice. ERT seems to be superior to

BMT at reducing GAGs in tissues provided that the immunological re-

sponses against the infused enzyme are low. ERT conferred an additive

effect to BMTat reducing GAGs in several visceral organs. Finally, pGAGs

are a better biomarker to monitor the therapeutic effect of MPS II.

Supplementary data to this article can be found online at http://dx.

doi.org/10.1016/j.ymgme.2013.09.013.

Conict of interest

T. Ohashi, H. Ida and Y. Eto have active research support from

Genzyme Japan Co., Ltd. and Shire Japan Co., Ltd. These activities have

been fully disclosed and are managed under a Memorandum of Under-

standing with the Conict of Interest Resolution Board of The Jikei

University School of Medicine.

Acknowledgments

Authors wish to thank Joseph Muenzer at University of NorthCarolina at Chapel Hill for providing us the MPS II mice, Hideto

Morimoto at JCR Pharmaceuticals Company Limited for providing us

antibody against human IDS and Genzyme Japan Co., Ltd. for providing

us Idursulfase. We also thank Seng Cheng at Genzyme Corporation

for critically reviewing our manuscript, Taku Sato at Tokyo Medical

and Dental University for helping us ow cytometry study and Sayoko

Iizuka and Eiko Kaneshiro at The Jikei University School of Medicine

for their excellent technical assistance. Finally we thank members of

Laboratory Animal Facility at The Jikei University School of Medicine

for helping us with the animal studies. This work was supported by

grant of the Vehicle Racing Commemorative Foundation.

References

[1] G. Bach, F. Eisenberg Jr., M. Cantz, E.F. Neufeld, The defect in the Hunter syndrome:deciency of sulfoiduronate sulfatase, Proc. Natl. Acad. Sci. U. S. A. 70 (1973)21342138.

[2] R. Martin, M. Beck, C. Eng, R. Giugliani, P. Harmatz, V. Munoz, J. Muenzer, Recogni-tion and diagnosis of mucopolysaccharidosis II (Hunter syndrome), Pediatrics 121(2008) e377e386.

[3] J. Muenzer, J.E.Wraith,M. Beck, R. Giugliani,P. Harmatz, C.M.Eng, A. Vellodi, R. Martin,U. Ramaswami, M. Gucsavas-Calikoglu, S. Vijayaraghavan, S. Wendt, A.C. Puga, B.Ulbrich, M. Shinawi,M. Cleary, D. Piper,A.M. Conway, A. Kimura, A phase II/IIIclinicalstudy of enzyme replacement therapy with idursulfase in mucopolysaccharidosis II(Hunter syndrome), Genet. Med. 8 (2006) 465473.

[4] T. Okuyama, A. Tanaka, Y. Suzuki, H. Ida, T. Tanaka, G.F. Cox, Y. Eto, T. Orii, JapanElaprase Treatment (JET) study: idursulfase enzyme replacement therapy in adultpatients with attenuated Hunter syndrome (Mucopolysaccharidosis II, MPS II),Mol. Genet. Metab. 99 (2010) 1825.

[5] J. Muenzer, M. Beck, C.M. Eng, R. Giugliani, P. Harmatz, R. Martin, U. Ramaswami, A.Vellodi, J.E. Wraith, M. Cleary, M. Gucsavas-Calikoglu, A.C. Puga, M. Shinawi, B.Ulbrich, S. Vijayaraghavan, S. Wendt, A.M. Conway, A. Rossi, D.A. Whiteman, A.

Kimura, Long-term, open-labeled extension study of idursulfase in the treatmentof Hunter syndrome, Genet. Med. 13 (2011) 95101.

[6] J.E. Wraith,M. Scarpa, M. Beck, O.A. Bodamer, L. De Meirleir,N. Guffon,A. MeldgaardLund, G. Malm, A.T. Van der Ploeg, J. Zeman, Mucopolysaccharidosis type II (Huntersyndrome): a clinical review and recommendations for treatment in the era ofenzyme replacement therapy, Eur. J. Pediatr. 167 (2008) 267277.

[7] S. Al Sawaf, E. Mayatepek, B. Hoffmann, Neurological ndings in Hunter disease:pathology and possible therapeutic effects reviewed, J. Inherit. Metab. Dis. 31 (2008)473480.

[8] Y. Sato, M. Fujiwara, H. Kobayashi, H. Ida, Massive accumulation of glycosaminogly-cans in the aortic valve of a patient with Hunter syndrome during enzyme replace-

ment therapy, Pediatr. Cardiol. (2013)(in press).[9] K. Wyatt, W. Henley, L. Anderson, R. Anderson, V. Nikolaou, K. Stein, L. Klinger, D.Hughes, S. Waldek, R. Lachmann, A. Mehta, A. Vellodi, S. Logan, The effectivenessand cost-effectiveness of enzyme and substrate replacement therapies: a longitudi-nal cohort study of people with lysosomal storage disorders, Health Technol. Assess.16 (2012) 1543.

[10] C. Peters, C.G. Steward, National Marrow Donor Program, International BoneMarrow Transplant Registry, Working Party on Inborn Errors, European BoneMarrow Transplant Group, Hematopoietic cell transplantation for inherited meta-bolic diseases: an overview of outcomes and practice guidelines, Bone MarrowTransplant. 31 (2003) 229239.

[11] J.J . Boelens, Trends in haematopoietic cell transplantation for inborn errors of me-tabolism, J. Inherit. Metab. Dis. 29 (2006) 413420.

[12] E.G. Shapiro, L.A. Lockman, M. Balthazor, W. Krivit, Neuropsychological outcomes ofseveral storage diseases with and without bone marrow transplantation, J. Inherit.Metab. Dis. 18 (1995) 413429.

[13] E.J. McKinnis, S. Sulzbacher, J.C. Rutledge, J. Sanders, C.R. Scott, Bone marrow trans-plantation in Hunter syndrome, J. Pediatr. 129 (1996) 145148.

[14] A. Vellodi, E. Young, A. Cooper, V. Lidchi, B. Winchester, J.E. Wraith, Long-term

follow-up following bone marrow transplantation for Hunter disease, J. Inherit.Metab. Dis. 22 (1999) 638648.

[15] N. Guffon, Y. Bertrand, I. Forest, A. Fouilhoux, R. Froissart, Bonemarrow transplanta-tion in children with Hunter syndrome: outcome after 7 to 17 years, J. Pediatr. 154(2009) 733737.

[16] A. Tanaka, T. Okuyama, Y. Suzuki, N. Sakai, H. Takakura, T. Sawada, T. Tanaka, T.Otomo, T. Ohashi, M. Ishige-Wada, H. Yabe, T. Ohura, N. Suzuki, K. Kato, S. Adachi,R. Kobayashi, H. Mugishima, S. Kato, Long-term efcacy of hematopoietic stem celltransplantation on brain involvement in patients with mucopolysaccharidosis typeII: a nationwide survey in Japan, Mol. Genet. Metab. 107 (2012) 513520.

[17] A.R. Garcia, J. Pan, J.C. Lamsa, J. Muenzer, The characterization of a murine modelof mucopolysaccharidosis II (Hunter syndrome), J. Inherit. Metab. Dis. 30 (2007)924934.

[18] T.Yokoi,H. Kobayashi,Y. Shimada,Y. Eto, N.Ishige, T. Kitagawa,M. Otsu, H. Nakauchi,H. Ida, T. Ohashi, Minimum requirement of donor cells to reduce the glycolipid stor-age following bone marrow transplantation in a murine model of Fabry disease,

J. Gene Med. 13 (2011) 262268.[19] T. Higuchi, H. Shimizu,T. Fukuda, S. Kawagoe,J. Matsumoto, Y. Shimada,H. Kobayashi,

H. Ida, T. Ohashi, H. Morimoto, T. Hirato, K. Nishino, Y. Eto, Enzyme replacementther-apy (ERT) procedure for mucopolysaccharidosis type II (MPS II) by intraventricularadministration (IVA) in murine MPS II, Mol. Genet. Metab. 107 (2012) 122128.

[20] R. Lawrence, J.R. Brown, K. Al-Mafraji, W.C. Lamanna, J.R. Beitel, G.J. Boons, J.D. Esko,B.E. Crawford, Disease-specic non-reducing end carbohydrate biomarkers formucopolysaccharidoses, Nat. Chem. Biol. 8 (2012) 197204.

[21] A. Rozdzynska, A. Tylki-Szymanska, A. Jurecka, J. Cieslik, Growthpattern andgrowthprediction of body height in children with mucopolysaccharidosis type II, ActaPaediatr. 100 (2011) 456460.

[22] T. Ohashi, S. Iizuka, Y. Shimada, T. Higuchi, Y. Eto, H. Ida, H. Kobayashi, Administra-tion of anti-CD3 antibodies modulates the immune response to an infusion ofalpha-glucosidase in mice, Mol. Ther. 20 (2012) 19241931.

[23] J.R. Hobbs, K. Hugh-Jones, A.J. Barrett, N. Byrom, D. Chambers, K. Henry, D.C. James,C.F. Lucas, T.R. Rogers, P.F. Benson, L.R. Tansley, A.D. Patrick, J. Mossman, E.P. Young,Reversal of clinical features of Hurler's disease and biochemical improvement aftertreatment by bone-marrow transplantation, Lancet 2 (1981) 709712.

[24] J. Muenzer, J.E. Wraith, L.A. Clarke, International Consensus Panel on Managementand Treatment of Mucopolysaccharidosis, Mucopolysaccharidosis I: managementand treatment guidelines, Pediatrics 123 (2009) 1929.

[25] D.A. Wolf, A.W. Lenander, Z. Nan, E.A. Braunlin, K.M. Podetz-Pedersen, C.B. Whitley,P. Gupta, W.C. Low, R.S. McIvor, Increased longevity and metabolic correctionfollowing syngeneic BMT in a murine model of mucopolysaccharidosis type I, BoneMarrow Transplant. 47 (2012) 12351240.

[26] A.A. Lau, H. Hannouche, T. Rozaklis, S. Hassiotis, J.J. Hopwood, K.M. Hemsley,Allogeneic stem cell transplantation does not improve neurological decits inmucopolysaccharidosis type IIIA mice, Exp. Neurol. 225 (2010) 445454.

[27] A. Langford-Smith, F.L. Wilkinson, K.J. Langford-Smith, R.J. Holley, A. Sergijenko, S.J.Howe, W.R. Bennett, S.A. Jones, J. Wraith, C.L. Merry, R.F. Wynn, B.W. Bigger, Hema-topoietic stem cell and gene therapy corrects primary neuropathology and behaviorin mucopolysaccharidosis IIIA mice, Mol. Ther. 20 (2012) 16101621.

[28] P.S. Kishnani, P.C. Goldenberg, S.L. DeArmey, J. Heller, D. Benjamin, S. Young, D. Bali,S.A. Smith, J.S. Li, H. Mandel, D. Koeberl, A. Rosenberg, Y.T. Chen, Cross-reactive im-munologic material status affects treatment outcomes in Pompe disease infants,Mol. Genet. Metab. 99 (2010) 2633.

[29] S.G. Banugaria, S.N. Prater, Y.K. Ng, J.A. Kobori, R.S. Finkel, R.L. Ladda, Y.T. Chen, A.S.Rosenberg, P.S. Kishnani, The impact of antibodies on clinical outcomes in diseasestreated with therapeutic protein: lessons learned from infantile Pompe disease,Genet. Med. 13 (2011) 729736.

http://dx.doi.org/10.1016/j.ymgme.2013.09.013http://dx.doi.org/10.1016/j.ymgme.2013.09.013http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0005http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0005http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0005http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0005http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0005http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0005http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0005http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0010http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0010http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0010http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0010http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0010http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0015http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0015http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0015http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0015http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0015http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0015http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0015http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0020http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0020http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0020http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0020http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0020http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0020http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0025http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0025http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0025http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0025http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0025http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0025http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0025http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0030http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0030http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0030http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0030http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0030http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0030http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0035http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0035http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0035http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0035http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0035http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0035http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0035http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0185http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0185http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0185http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0045http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0045http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0045http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0045http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0045http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0045http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0045http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0050http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0050http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0050http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0050http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0050http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0050http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0050http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0055http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0055http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0055http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0055http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0060http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0060http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0060http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0060http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0060http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0065http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0065http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0065http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0065http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0070http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0070http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0070http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0070http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0070http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0075http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0075http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0075http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0075http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0075http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0080http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0080http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0080http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0080http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0080http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0080http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0080http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0080http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0080http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0085http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0085http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0085http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0085http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0085http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0090http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0090http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0090http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0090http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0090http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0090http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0095http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0095http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0095http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0095http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0095http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0095http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0100http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0100http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0100http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0100http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0100http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0100http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0100http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0105http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0105http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0105http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0105http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0105http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0110http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0110http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0110http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0110http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0110http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0115http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0115http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0115http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0115http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0115http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0115http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0120http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0120http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0120http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0120http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0120http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0125http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0125http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0125http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0125http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0125http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0125http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0130http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0130http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0130http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0130http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0130http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0130http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0130http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0135http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0135http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0135http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0135http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0135http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0135http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0140http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0140http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0140http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0140http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0140http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0140http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0145http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0145http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0145http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0145http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0145http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0145http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0145http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0145http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0145http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0145http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0140http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0140http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0140http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0140http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0135http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0135http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0135http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0135http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0130http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0130http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0130http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0125http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0125http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0125http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0125http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0120http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0120http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0120http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0115http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0115http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0115http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0115http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0110http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0110http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0110http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0105http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0105http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0105http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0100http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0100http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0100http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0095http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0095http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0095http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0095http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0090http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0090http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0090http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0090http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0085http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0085http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0085http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0080http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0080http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0080http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0080http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0080http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0075http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0075http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0075http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0070http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0070http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0070http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0065http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0065http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0060http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0060http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0060http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0055http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0055http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0050http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0050http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0050http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0050http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0050http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0045http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0045http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0045http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0045http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0045http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0185http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0185http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0185http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0035http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0035http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0035http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0030http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0030http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0030http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0030http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0025http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0025http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0025http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0025http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0025http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0020http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0020http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0020http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0020http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0015http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0015http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0015http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0015http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0015http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0010http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0010http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0010http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0005http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0005http://refhub.elsevier.com/S1096-7192(13)00325-9/rf0005http://dx.doi.org/10.1016/j.ymgme.2013.09.013http://dx.doi.org/10.1016/j.ymgme.2013.09.013


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[30] C.B.Whitley, J.R. Utz, MaroteauxLamy syndrome (mucopolysaccharidosis type VI):a single dose of galsulfase further reduces urine glycosaminoglycans after hemato-poietic stem cell transplantation, Mol. Genet. Metab. 101 (2010) 346348.

[31] Y.B. Sohn, S.W. Park, S.H. Kim, S.Y. Cho, S.T. Ji, E.K. Kwon, S.J. Han, S.J. Oh, Y.J. Park,A.R. Ko, K.H. Paik, J. Lee, D.H. Lee, D.K. Jin, Enzyme replacement therapy improves

joint motion and outcome of the 12-min walk test in a mucopolysaccharidosistype VI patient previously treated with bone marrow transplantation, Am. J. Med.Genet. A 58A (2012) 11581163.

[32] S.S. Grewal, R. Wynn, J.E. Abdenur, B.K. Burton, M. Gharib, C. Haase, R.J. Hayashi, S.Shenoy, D. Sillence, G.E. Tiller, M.E. Dudek, A. van Royen-Kerkhof, J.E. Wraith, P.Woodard, G.A. Young, N. Wulffraat, C.B. Whitley, C. Peters, Safety and efcacy of

enzyme replacement therapy in combination with hematopoietic stem cell trans-plantation in Hurler syndrome, Genet. Med. 7 (2005) 143146.[33] J. Cox-Brinkman, J.J. Boelens, J.E. Wraith, A. O'meara, P. Veys, F.A. Wijburg, N.

Wulffraat, R.F. Wynn, Haematopoietic cell transplantation (HCT) in combinationwith enzyme replacement therapy (ERT) in patients with Hurler syndrome, BoneMarrow Transplant. 38 (2006) 1721.

[34] J. Tolar, S.S. Grewal, K.J. Bjoraker, C.B. Whitley, E.G. Shapiro, L. Charnas, P.J.Orchard, Combination of enzyme replacement and hematopoietic stem cell trans-plantation as therapy for Hurler syndrome, Bone Marrow Transplant. 41 (2008)531535.

[35] R.F. Wynn, J. Mercer, J. Page, T.F. Carr, S. Jones, J.E. Wraith, Use of enzyme re-placement therapy (Laronidase) before hematopoietic stem cell transplantationfor mucopolysaccharidosis I: experience in 18 patients, J. Pediatr. 154 (2009)135139.

[36] D. Sillence,K. Waters, S.Donaldson, P.J. Shaw, C. Ellaway, Combinedenzyme replace-menttherapyand hematopoietic stemcell transplantationi n mucopolysaccharidosistype VI, JIMD Rep. 2 (2012) 103106.

[37] P.I. Dickson, N.M. Ellinwood, J.R. Brown, R.G. Witt, S.Q. Le, M.B. Passage, M.U. Vera,B.E. Crawford, Specic antibody titer alters the effectiveness of intrathecal enzymereplacement therapy in canine mucopolysaccharidosis I, Mol. Genet. Metab. 106(2012) 6872.

[38] R. Lawrence, J.R. Brown, F. Lorey, P.I. Dickson, B.E. Crawford, J.D. Esko, Glycan-basedbiomarkers for mucopolysaccharidoses, Mol. Genet. Metab. 111 (2013) 7383.

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Akiyama 2014. Enzyme Augmentation Therapy Enhances the Therapeutic Efficacy of Bone Marrow Transplantation in Mucopolysaccharidosis Type II Mice