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1
* Department of Human Genetics and
Institute for Gene Therapy and Molecular Medicine, Mount Sinai School of Medicine, New York, New York 10029, USA; and
Department of Biochemistry, Hebrew University-Hadassah School of Medicine, Jerusalem, Israel 92710
1Correspondence: Department of Human Genetics, Mount Sinai School of Medicine, 1425 Madison Ave., 14–20A, New York, NY 10029, USA. E-mail: Schuchman{at}msvax.mssm.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: lysosomal storage disease • enzyme therapy • animal models
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2)
.
Type A NPD presents with failure to thrive, hepatosplenomegaly, and
progressive neurodegeneration that generally leads to death by three
years of age. In contrast, patients with Type B NPD develop
hepatosplenomegaly and/or pulmonary insufficiency, but usually have
little neurological involvement and survive into adolescence or
adulthood (3)
. ASM is the enzyme required to hydrolyze sphingomyelin (SPM) into ceramide and phosphocholine. In most normal tissues, SPM constitutes from 5 to 20% of the total cellular phospholipid, but in NPD patients these levels may be elevated up to over 70%. It is presumed that both forms of NPD are caused by the accumulation of SPM in various cell types (e.g., macrophages, neurons), leading to impairment of their normal cellular function. However, the precise nature of the phenotypic variation in NPD is not understood.
The rationale underlying enzyme replacement therapy (ERT) for the
treatment of lysosomal storage disorders derives from early cell
culture studies showing that cells from patients affected with these
disorders could be metabolically ‘corrected’ when they were cultured
together with either normal cells or conditioned media obtained from
normal cells (4)
. Subsequently, it was found that the
‘cross-correcting factors’ present in normal cell media were
lysosomal enzymes that were being released from the cells at low levels
(5)
. Early attempts at ERT in human patients with
lysosomal disorders were unsuccessful (6
7
8
9
10
11)
, primarily
because of an inadequate understanding of receptor-mediated endocytosis
and insufficient sources of highly purified enzymes. These limitations
have now been largely overcome, and ERT has proved clinically
successful for at least one lysosomal storage disorder: Type 1 Gaucher
disease (12
13
14
15)
.
ERT for NPD became possible after the cloning of the human ASM cDNA and
gene (16
17
18)
, the construction of mouse models deficient
in ASM activity (19
, 20)
, and the creation of a Chinese
hamster ovary (CHO) cell line overexpressing recombinant human ASM
(rhASM) (21)
. The ASM-deficient (ASMKO) mouse models have
clinical, biochemical, and pathological findings similar to those seen
in human NPD patients, making them valuable tools for the investigation
of disease pathogenesis and evaluation of therapeutic approaches.
Relevant to the success of ERT for NPD, it has recently been shown that
ASM is released from normal cells at high levels (22
, 23)
.
Intriguingly, this secreted form of ASM (also known as sSMase) required
zinc cations to become activated, explaining why it had not been
previously detected by standard enzyme assay systems, which generally
were carried out in the presence of EDTA. In stark contrast to the
secreted enzyme, the intracellular enzyme is highly active at low pH in
the absence of exogenous zinc or other divalent cations. The secreted
enzyme can be taken up by NPD cells via a receptor-mediated processes
that delivers it to the lysosomes; within lysosomes, the internalized
enzyme comes in contact with zinc and is activated.
As part of our ongoing efforts to develop an effective treatment for
Types A and B NPD, the present study evaluated the efficacy of ERT
using the ASMKO mouse model and compared these results to those
obtained previously from bone marrow transplantation (BMT) (24
, 25)
and hematopoietic stem cell gene therapy (HSCGT) (S. R. P. Miranda et al. unpublished results). The data presented
document the efficacy of ERT for the treatment of non-neurological Type
B NPD and highlight its limitations for the treatment of neurological
Type A NPD.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Affected mice have no detectable ASM
activity, but develop normally until 
8 wk of age, when ataxia and
mild tremors become noticeable. The disease then follows a
neurodegenerative course that leads to death between 6 and 8 months of
age. Characteristic lipid-laden foam cells (NPD cells) are found in
most major organs, associated with elevated SPM levels. Homozygous
(-/-) mice are distinguished from normal (+/+) or heterozygous (+/-)
animals using a polymerase chain reaction assay (19)
. The
animals were maintained on a 12 h light/dark cycle with water
ad libitum and Purina rodent chow 5001.
Production and purification of recombinant human ASM
The eukaryotic expression vector p91023 was used to stably
overexpress active human ASM in a CHO cell line (21)
.
Briefly, this overexpression system relied on coamplification of the
human ASM cDNA and the adjacent dihydrofolate reductase gene in the
presence of methotrexate (26
, 27)
. Stable amplification of
the human ASM cDNA led to overexpression and secretion of rhASM. A
clonal CHO cell population overexpressing rhASM was obtained by
methotrexate amplification, adapted to suspension cultures, and
maintained in a CELLMAX 100 bioreactor system (21)
. The
CELLMAX 100 system enabled collection of 1 l of culture media/week
for enzyme purification. The purification method resulted in the
isolation of rhASM in an essentially homogeneous form
(21)
.
Enzyme administration
Three groups of test animals were injected with purified rhASM.
Initially, doses of 0.3, 1, 3, and 10 mg rhASM/kg of mouse body weight
were administered intravenously (i.v.) (50 µl injection volume) into
adult (5 month) ASMKO mice for 2 wk (4 mice each per dose). Mice
received injections every other day during the 2 wk period (7
injections) and were killed 48 h after the last injection to
assess their tissue SPM content (described below). Based on these
results, three additional 5-month-old ASMKO animals were then injected
i.v. with 5 mg/kg every other day for 8 days (4 injections) and killed
for histological analysis on day 10 (see below). For long-term
analysis, 1 mg/kg of rhASM was administered i.v. once a week for 15 wk
into 10 ASMKO animals, starting when the animals were 3 wk old. Five
animals each were used for biochemical or histological analysis.
Immediately before each injection the enzyme was filtered using a 0.2
µm filter.
ELISA and immunoprecipitation assays
To detect the presence of antibodies against injected rhASM,
enzyme-linked immunoassays (ELISA) were performed on plasma from
treated and untreated mice. Purified rhASM (1 µg) was incubated for
18 h in 96-well microtiter plates. The wells were washed 3x with
washing buffer 1 (WB1: 10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.05% Tween
20) and then blocked for 1 h with 3% bovine serum albumin (BSA)
in phosphate-buffered saline (PBS) at room temperature. Dilutions of
treated and untreated mouse plasma in WB1 (100 µl of
10-2, 10-3, or
10-4 dilutions) were added to the wells and
incubated at 37°C for 3 h. The wells were then washed 3x with
WB1, followed by the addition of 100 µl of peroxidase-conjugated goat
anti-mouse IgG (Sigma, St. Louis, Mo.) (1:500 dilution in WB1) and a
2 h incubation at room temperature. The wells were again washed
3x with WB1, then twice more with washing buffer 2 (10 mM Tris-HCl pH
7.5, 150 mM NaCl); 100 µl of peroxidase substrate (‘ready-to-use’
peroxidase substrate for ELISA-ABTS solution; Boehringer Mannheim,
Mannheim, Germany) was added to the wells and incubated at room
temperature for 30 min. An automated ELISA plate reader was used to
determine the absorbance at 405 nm.
Immunoprecipitation assays were used to assess whether the antibodies
present in the treated mice were inactivating the injected enzyme. A
solution of 500 U (1 µg) of rhASM, 1 ml of PBS, 10 µl of mouse
plasma, and 1 mg/ml BSA was incubated for 18 h at 4°C on a
rotating wheel; 100 µl of protein G-Sepharose diluted 1:1 in PBS was
then added to the solution and incubated for 4 h at 4°C. After
centrifugation at 14000 rpm for 2 min, supernatants and pellets were
assayed for ASM activity.
ASM activity assay
SPM covalently linked to the fluorescent probe BODIPY (BODIPY
dodecanoyl sphingosyl phosphocholine; BOD12-SPM) was synthesized as
described previously for lissamine rhodamine SPM (LR12-SPM)
(28)
, except that BODIPY dodecanoic acid (Molecular Probes
Inc., Eugene, Oreg.) was condensed with sphingosyl phosphocholine.
Cleavage of BOD12-SPM by ASM releases fluorescent ceramide, which can
be quantified after separation by TLC or organic extraction. WBCs or
tissues from the treated or control mice were obtained as described
previously (25)
and homogenized in 0.2% Triton X-100 on
ice using three 10 s bursts of a Potter-Elvehjem tissue
homogenizer (Thomas Scientific, Swedesboro, N.J.). Total protein was
determined by the method of Stein et al. (29)
. The
standard 15 µl ASM assay mixture consisted of 10 µl of sample
(homogenized cells or tissues) and 2 nmol of BOD12-SPM suspended in 0.1
M sodium acetate buffer, pH 5.2, containing 0.6% Triton X-100 and
either 5 mM EDTA (for detection of the non-zinc-dependent ASM activity)
or 0.1 mM ZnCl2 (for detection of the
zinc-stimulated ASM activity) (23)
. After incubating the
assay mixture at 37°C (up to 3 h), the samples were loaded onto
TLC plates (LK6 D Silica gel 60, Whatman, Clifton, N.J.) and resolved
using chloroform/methanol (95:5 v/v). After resolution, the band
containing the fluorescently labeled ceramide was scraped from the TLC
plates, extracted in chloroform/methanol/water (1:2:1 v/v) for 15 min
at 55°C, and quantified in a spectrofluorometer (fluorescence
spectrophotometer 204-A; Perkin Elmer, Norwalk, Conn.). The excitation
and emission settings were 505 and 530 nm, respectively.
Sphingomyelin analysis
SPM levels were determined from the phosphate content in each
sample (30)
. Tissues (brain, liver, lung, spleen, kidney,
and heart) were homogenized in chloroform/methanol (1:2 v/v) for lipid
extraction. Alkaline phospholipid hydrolysis was performed on the
samples in 0.4 N KOH/90% methanol for 2 h at 55°C. To avoid
interference from plasmalogen and other phospholipids in the brain
extracts, acidic hydrolysis was carried out on these samples using 0.6
M HCl in ethanol for 30 min at 37°C prior to the alkaline hydrolysis.
After hydrolysis, the samples were incubated with perchloric acid
(70%) for 40 min at 180°C, followed by treatment with 0.5% ammonium
molybdate and Fiske & Subbarow reducer (4 mg/ml; Sigma) for 10 min at
100°C. Absorbance at 830 nm was measured in a spectrophotometer
(Model 1201, Milton Roy Spectronic Inc. Rochester, N.Y.).
Histopathological studies
Mice were anesthetized with ketamine (Sigma; 0.5 g/kg of body
weight) and subjected to cardiac perfusion. An incision was made in the
right atrium to allow blood to flow out, and a cannula was introduced
through the left ventricle into the aorta, delivering 50 ml of warm
0.9% NaCl solution. Tissue samples for light microscopy were fixed in
buffered 10% formalin, paraffin-embedded, sectioned, and stained with
hematoxylin and eosin.
Behavioral studies
Motor coordination tests were conducted using a rota-rod
treadmill for mice (7650 Accelerating model, Ugo Basile Biological
Research Apparatus, Italy) (31)
. In this apparatus, a
motor sets the rotor in motion via the gear belt at a selected speed.
When the mouse falls off its cylinder section, the plate below trips
and the corresponding counter is disconnected, thereby recording the
animal’s endurance time in seconds. By subjecting animals to an
accelerating drum, screening results are less scattered
(32)
. The machine was set to an initial speed of 32 rpm
and the acceleration was increased 1x every 25–30 s. Treated mice
were analyzed at 4 months of age (after the 10th injection) along
with ASMKO and normal control animals. Their scores were registered in
2 consecutive days, carefully keeping the conditions in each test as
similar as possible. Three tests were performed each day, with a rest
time of 1 h between trials. A maximum time limit of 360 s/test was
established.
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RESULTS |
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. To further evaluate the effects of ERT in ASMKO
mice, a total of 16 five-month-old ASMKO mice were injected with 0.3,
1, 3, or 10 mg/kg of rhASM every other day for 14 days (7 injections, 4
animals per dose). The animals were killed 48 h after the last
injection and their tissues were analyzed for SPM content. The mice
used in these studies had clinical and pathological disease at the
onset of ERT, including ataxia, tremors, elevated SPM levels in their
organs, and foam cell infiltrates. Thus, these studies were designed to
evaluate whether accumulated substrate could be depleted in diseased
mice after short-term ERT. Figure 1
shows the reduction in SPM content in different tissues of the treated
animals. Note that a substantial SPM reduction occurred in the livers,
spleens, and hearts, whereas only a partial reduction was observed in
the lungs at the highest enzyme dose (10 mg/kg). Essentially no
alterations in the SPM contents were observed in the kidneys and
brains. Next, a group of three additional 5-month-old ASMKO mice
received i.v. injections of rhASM (5 mg/kg) every other day for 8 days
(four injections), and then were killed for histological analysis on
day 10. Note the widespread infiltration of lipid-filled foam cells
(indicated by the white open spaces) throughout the livers and spleens
of 5-month-old ASMKO animals and the almost complete occlusion of the
airway spaces in the lungs (Fig. 2
). Also note the marked improvement in the livers, spleens, and lungs of
the animals treated by short-term ERT.
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Long-term ERT
Based on the encouraging results of short-term ERT in older ASMKO
animals (see above), a long-term ERT (15 wk) protocol was then carried
out on 10 ASMKO mice using an intermediate dose of enzyme (1 mg/kg)
injected once per week. In contrast to the short-term protocols, these
recipients were 3 wk of age and presymptomatic at the time ERT was
initiated.
Figure 3
depicts the SPM results obtained from the long-term protocol. The data
demonstrate that ERT prevented significant SPM accumulation in all
organs analyzed, with the exception of the brain. Figure 4
shows representative histological analysis of tissue sections from
several organs obtained from treated ASMKO and control mice. Note that
by 5 months of age, untreated ASMKO mice exhibited massive lipid
storage as compared to normal animals. Numerous foam cells interspersed
throughout the liver and spleen sections were observed along with
extensive infiltration of alveoli and septa in the lungs, occluding the
airways. Notably, tissues from the treated ASMKO animals had an almost
normal histological appearance.
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Analysis of brain sections from the treated ASMKO mice revealed massive
Purkinje cell drop-out equivalent to that observed in untreated ASMKO
mice (not shown), suggesting that the injected rhASM did not cross the
blood–brain barrier to a significant degree. Consistent with these
results, all of the treated mice developed severe ataxia, exhibited
behavioral abnormalities (see below), and died at about the same age as
untreated ASMKO mice (average age of death 6 months, not shown).
Adverse reactions and immune responses
Most of the ASMKO mice treated with rhASM showed no adverse
reactions. However, 3 of the 10 animals in the long-term study died
immediately after the injections. The cause of death was likely related
to restraining, anesthesia, or the injection procedure itself. No
anti-anaphylactic drugs were administered before the injections and no
acute immune reactions were observed. Serum samples were collected
every other week from the 7 remaining mice beginning 2 wk after the
first enzyme injection. ELISA analyses were carried out to detect the
presence of anti-rhASM antibodies and immunoprecipitation was used to
determine whether such antibodies inactivated the enzyme activity. All
of the treated animals had measurable levels of anti-hASM antibodies by
the 5th injection (Fig. 5
), but these antibodies did not inactivate the enzyme as determined by
immunoprecipitation (not shown).
|
Behavioral assessment
Rota-rod performance was used to assess the motor behavior of
treated mice in comparison with normal and affected control animals.
Figure 6
summarizes the average scores from six rota-rod trials performed on two
consecutive days at 4 months of age (i.e., after the 10th enzyme
injection). Note that the treated animals had average scores, which
were not significantly different from the ASMKO control animals,
consistent with the lack of histological or biochemical improvement in
the central nervous system (CNS).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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34
35
), leading to their large-scale
purification. A similar strategy was used to overexpress and purify
rhASM (21)
. The physical and kinetic properties of the
purified rhASM were nearly identical to those of enzyme purified from
human urine, providing a readily available source of active enzyme for
the evaluation of ERT in the ASMKO mice.
Our initial ERT studies revealed that the T1/2 of
rhASM after a single i.v. injection into ASMKO mice was 2 min and
that >90% of the injected enzyme was taken up by the liver
(21)
. These studies also showed that the majority of the
internalized enzyme was delivered to lysosomes and that 30–50% of
the enzyme uptake by cells was dependent on the mannose-6-phosphate
receptor system.
The present study extends these findings and reports biochemical, histological, and clinical analysis of ERT in the ASMKO mice. The major organ sites of pathology in NPD are the liver, spleen and lung, as well as the brain in Type A NPD. Thus, our analysis was focused on these four organs. Initially, a short-term protocol was carried out in which 5-month-old, symptomatic ASMKO mice were injected with varying doses (ranging from 0.1 to 10 mg/kg) of rhASM every other day for 14 days. These studies revealed that the SPM levels in the livers and spleens (as well as the hearts) of the treated animals were markedly reduced as compared to age-matched ASMKO control animals, suggesting that the disease phenotype may be reversible. The SPM levels in the lungs were only modestly reduced at the highest enzyme levels (10 mg/kg), perhaps due to the reduced vascularization of this tissue as compared to the other three. No substrate reduction was observed in brains (or kidneys) from these short-term studies. Histological analysis of 5-month-old ASMKO mice treated with only four injections (5 mg/kg) of rhASM confirmed these biochemical results and revealed that the extent of histological reversal in the lungs was even greater than that expected from the biochemical studies.
We then treated a group of 3 wk old, presymptomatic ASMKO animals with
weekly rhASM injections of 1 mg/kg for 15 wk. As expected, anti-hASM
antibodies were detected in the plasma of treated mice by the 5th
injection, but immunoprecipitation analysis demonstrated that these
antibodies were non-neutralizing. SPM levels determined at the end of
the 15 wk treatment period revealed that the group receiving ERT had
markedly lower substrate levels than untreated, age-matched ASMKO
control animals. This was most evident in the hearts, spleens, and
livers, but was also observed in the lungs and to a very limited degree
in the kidneys. The only tissue for which there was no evidence of
substrate reduction was the brain, consistent with the fact that by the
end of the treatment schedule (i.e., when the animals were 18 wk of
age), all of the ASMKO mice receiving ERT had severe ataxia and
neurological disease. Histological analysis again corroborated the
biochemical results and showed a marked improvement in the
reticuloendothelial organs of the ERT treatment group as opposed to
untreated ASMKO controls.
It is of interest to compare these ERT results to previous results from
our group using the ASMKO mice to evaluate two other therapeutic
approaches, BMT and HSCGT (24
, 25
; S. R. P.
Miranda et al., unpublished results). The most noticeable difference
between BMT or HSCGT vs. ERT is in the effects on CNS disease. ERT led
to essentially no improvements in CNS disease, whereas BMT and HSCGT
slowed the progression of the CNS disease but did not prevent it. These
latter results were obtained even though very few hematopoietically
derived cells crossed the blood–brain barrier and no increases in ASM
activity were detected in the treated brain homogenates (24
, 25)
. Thus, the CNS disease in ASMKO mice is responsive to very
low levels of ASM activity and the lack of a similar response by ERT is
likely due to the fact that essentially no rhASM was able to cross the
blood–brain barrier after i.v. injection. Other than the CNS effects,
the therapeutic response of the ASMKO mice to ERT were similar to BMT
or HSCGT, although these latter approaches seemed to be modestly more
effective in treating the lung pathology. Responses in the livers and
spleens were similar. One of the most notable results obtained from the
ERT study was that as few as four injections of rhASM into older ASMKO
mice could significantly reverse substrate accumulation in ASMKO mice.
Since the BMT or HSCGT protocols were only carried out on
presymptomatic animals, these results could not be directly compared.
It is also of interest to compare our ERT results to those obtained in
animal models of other lysosomal storage diseases. For example, in the
murine model of mucopolysaccharidosis (MPS) type VII, ERT initiated
early in life led to marked improvements in visceral organ
glycosaminoglycan storage and partial improvements in learning, memory,
and hearing deficits (36)
. ERT in the ASMKO mice also led
to marked improvements in visceral organ storage, but no detectable
neurological changes. It must be recognized, however, that the tests
used to assess neurological changes in the two studies (e.g., cognitive
in the MPS VII mice vs. motor coordination in the ASMKO mice) were
different, perhaps explaining the differing results. In addition, the
ERT treatment in the MPS VII mice was initiated slightly earlier than
in the ASMKO mice, perhaps before complete closure of the blood–brain
barrier. In avian and feline models of Pompe’s disease and MPS type
VI, respectively (37
, 38)
, ERT initiated at various ages
also led to variable amounts of visceral organ pathological changes and
clinical improvement, again suggesting that ERT is an effective way to
treat many of these disorders. Similar studies have been carried out in
a dog model of MPS type I (39)
and several other lysosomal
disease models. Although a systematic assessment of age dependency has
not been carried out in most cases, in general, the results clearly
indicate that ERT should be beneficial for these disorders and that the
earlier therapy is initiated, the better the clinical response.
In summary, the data presented here indicate that ERT is likely to be a useful therapeutic approach for non-neurological, Type B NPD patients and that even in older patients in which the SPM levels are markedly elevated, there may be some therapeutic response. The major organ sites of pathology in Type B NPD are the liver, spleen, and lung, three sites that showed a strong response to ERT in the mouse model. For Type A NPD, ERT is unlikely to evoke any therapeutic response in the CNS, limiting its usefulness for the treatment of this disorder.
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ACKNOWLEDGMENTS |
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Received for publication March 2, 2000.
Revision received April 14, 2000.
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-galactosidase A results in its aggregation, crystallization in lysosomes, and selective secretion. J. Cell Biol. 119,1137-1150This article has been cited by other articles:
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  S. Schroder, F. Matthes, P. Hyden, C. Andersson, J. Fogh, S. Muller-Loennies, T. Braulke, V. Gieselmann, and U. Matzner Site-specific analysis of N-linked oligosaccharides of recombinant lysosomal arylsulfatase A produced in different cell lines Glycobiology, February 1, 2010; 20(2): 248 - 259. [Abstract] [Full Text] [PDF]
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 S. Uhlig and E. Gulbins Sphingolipids in the Lungs Am. J. Respir. Crit. Care Med., December 1, 2008; 178(11): 1100 - 1114. [Abstract] [Full Text] [PDF]
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M. S. Sands, C. A. Vogler, K. K. Ohlemiller, M. S. Roberts, J. H. Grubb, B. Levy, and W. S. Sly Biodistribution, Kinetics, and Efficacy of Highly Phosphorylated and Non-phosphorylated beta -Glucuronidase in the Murine Model of Mucopolysaccharidosis VII J. Biol. Chem., November 9, 2001; 276(46): 43160 - 43165. [Abstract] [Full Text] [PDF]
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