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Blood, Vol. 95 No. 11 (June 1), 2000:
pp. 3631-3633
BRIEF REPORT
Reduction of lysosomal storage in murine mucopolysaccharidosis
type VII by transplantation of normal and genetically modified
macrophages
Toya Ohashi,
Takashi Yokoo,
Sayoko Iizuka,
Hiroshi Kobayashi,
William S. Sly, and
Yoshikatsu Eto
From the Department of Gene Therapy, Institute of DNA
Medicine, Jikei University School of Medicine, Tokyo, Japan;
Department of Pediatrics, Jikei University School of Medicine, Tokyo,
Japan; Department of Medicine II, Jikei University School of Medicine,
Tokyo, Japan; Edward A. Doisy Department of Medicine/Cardiology, and
Molecular Biology, St Louis University School of Medicine, St
Louis, MO.
 |
Abstract |
This study examined the ability of macrophages to serve as target
cells of gene therapy for mucopolysaccharidosis (MPS) type VII using a
murine model. Bone marrow cells were harvested from syngeneic normal
mice and differentiated to macrophages. These cells were given to
nonmyeloablated MPS VII mice. After transplantation, donor cells
populated the liver and spleen. The pathologic improvement at day 38 after transplantation was significant and glycosaminoglycan storage was
reduced. To develop gene therapy using this system, a retroviral vector
expressing human  -glucuronidase (HBG) was used to infect macrophages
cultivated from MPS VII mice and given to nonmyeloablated MPS VII mice.
At 38 days after transplantation, HBG-positive cells were still
observed histochemically and pathologic improvement was significant.
These observations suggest that macrophage transplantation is a
promising method for treatment of murine MPS VII without myeloablation,
and macrophages may be good target cells for ex vivo gene therapy for
MPS VII.
(Blood. 2000;95:3631-3633)
© 2000 by The American Society of Hematology.
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Introduction |
Mucopolysaccharidosis type VII (MPS VII), which is also
known as Sly syndrome, is a lysosomal storage disease caused by
deficiency of human -glucuronidase (HBG). This results in
accumulation of glycosaminoglycans (GAGs) in various
tissues.1 Murine models of MPS VII are available, which
exhibit biochemical and clinical phenotypes similar to those of the
human disease.2,3 Using this model, various gene therapy
approaches, including gene transfer to hematopoietic stem cells (HSC),
have been reported.4 So far, gene transfer to HSC appears
the most practical gene therapy approach to treat lysosomal storage
disease, including MPS VII. However, gene transfer to human HSC by
various gene transfer methods has been inefficient. In a clinical trial
of gene therapy for a lysosomal storage disease based on transferring
the therapeutic gene to human HSC, the transduction efficiency was too
low to alter the disease phenotype.5 To overcome the
problems of gene transfer to HSC, we studied the usefulness of
macrophages as target cells for MPS VII gene therapy.
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Materials and methods |
Mice
Breeding pairs of (+/ ) mice (B6.C-H2bml/BY
Bir-gusmps/+) were purchased from the Jackson
Laboratory (Bar Harbor, ME) and bred. Homozygous mutants
(/), heterozygous (+/), and wild-type (+/+) animals were identified by DNA analysis.6,7
Macrophage culture
Macrophages from bone marrow cells were cultivated from normal mice
(+/+) and MPS VII mice (/) as described.8,9
Briefly, bone marrow cells were harvested from femoral bones of mice
and seeded onto unprocessed 100-mm polystyrene dishes
(2 × 105 cells/dish) in 50% Dulbecco's modified
Eagle's medium, 10% fetal calf serum, 20% horse serum, and 20% L929
conditioned medium (macrophage medium). Two weeks after the initiation
of culture, the adherent cells were collected and suspended in
phosphate-buffered saline. We usually obtained
3.0 × 106 adherent cells from one 100-mm dish
(originally seeded at 2 × 105 bone marrow cells).
More than 95% of the adherent cells were positive for macrophage
markers (CD18, CD11b, F4/80 antigen) (data not shown).
Transduction of macrophages by retroviral vector (MFG-HBG)
One day before harvesting of bone marrow cells, the medium from
retroviral producer cells was changed to macrophage medium. The
retroviral vector has been described in detail
previously.10 The next day, the bone marrow cells of MPS
VII mice (/) were harvested and
2 × 105 bone marrow cells were suspended in 7 mL of
filtered (0.45 µm pore size) macrophage medium conditioned by
retroviral vector-producer cells containing 8 µg/mL of polybrene and
plated in 100-mm unprocessed dishes. The next day, 4 mL of medium
containing nonadherent cells was removed and mixed with 4 mL of
filtered macrophage medium conditioned by retroviral vector-producer
cells. Polybrene was added at final concentration of 8 µg/mL. The
mixture (total 8 mL) was centrifuged at 2400g for 2 hours at
4°C. After centrifugation, 4 mL of medium was discarded without
disturbing the cell pellet; then the cells were suspended and added
back to dishes. This method was repeated for 5 consecutive days, then
the culture was continued for 2 weeks. Just before transplantation into
mice, HBG gene expression was determined by HBG activity assay and
reverse transcriptase-polymerase chain reaction (RT-PCR).
Transplantation to MPS VII mice
Aliquots of 3.6 × 106 normal macrophages or
genetically modified macrophages were infused intravenously into 8- to
10-week-old nonmyeloablated MPS VII mice. The results of our
preliminary studies indicated that the mice tolerated this number of
cells well. Tissues were isolated for analysis at 7 or 38 days after transplantation.
Histologic and biochemical studies
The activity of HBG in the liver and spleen was also assayed
histochemically.7 Thin sections (0.5 µm) of tissue were
stained with toluidine blue to evaluate lysosomal storage.7
HBG activity in the tissues was assayed as described.11 The
concentration of GAGs in the liver and spleen was measured as
described.12
| |
Results and discussion |
Human HSC are resistant to retoroviral infection.5 To
seek an alternative approach for the treatment of MPS VII, we used macrophages as target cells for gene transfer instead of HSC.
Recently, Kennedy et al13 demonstrated that murine
macrophages cultured in vitro can enter tissues and engraft after
transplantation. Moreover, Hahn et al14 demonstrated that
expression of the therapeutic gene in macrophage lineage cells was
therapeutic in a mouse galactosialidosis model. These observations
supported our strategy for treatment of MPS VII by transplantation of
normal or genetically modified macrophages. At 7 days after injection
of normal macrophages into nonmyeloablated MPS VII mice, the HBG
activities in the liver and spleen were increased (Table 1) from
0.84 ± 0.75 to 32.9 ± 8.6 nmol/h/mg and from
0.44 ± 0.39 to 35.0 ± 22.3 nmol/h/mg, respectively. Histochemical analysis of HBG activity after transplantation indicated that many enzyme-competent macrophages entered the liver and spleen (data not shown). In contrast to the liver and spleen, increases in
enzymatic activities in other tissues such as the brain, lung, kidney,
and heart were minimal (data not shown). Although the HBG activity in
the liver and spleen subsequently fell to 3.6 ± 1.5 and
2.3 ± 0.6 nmol/h/mg, respectively, by 38 days, a number of
HBG-positive cells were still observed histochemically and pathologic
improvement was significant. Light micrographs of the liver and spleen
at day 38 are shown in Figure 1. The
abundant lysosomal storage in Kupffer cells was reduced in treated
animals, with small amounts of storage still seen in hepatocytes
(Figure 1B). In the spleen, the abundant lysosomal storage in red pulp was also reduced (Figure 1E).
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| Fig 1.
Live and spleen at day 38 after transplantation.
The liver of untreated, age-matched MPS VII mice (A) showed lysosomal
distention in Kupffer cells and a small amount of storage in
hepatocytes. At day 38 after transplantation of normal macrophages (B),
Kupffer cell storage was markedly reduced. However, reduction of
storage in hepatocytes was less marked. The untreated MPS VII mouse
spleen showed abundant lysosomal storage in sinus-lining cells in the
red pulp (D). At day 38 after transplantation of normal macrophages,
there was a similar marked reduction in the amount of lysosomal storage
(E). Thirty-eight days after transplantation of genetically modified
macrophages, histologic findings in the liver and spleen were similar
to those of mice that received normal macrophages (C and F) (A-F,
toluidine blue; original magnification × 200).
|
|
We analyzed the amounts of various GAGs in the liver and spleen to
confirm the histologic data. Although levels of most of the GAGs
analyzed were reduced in both the liver and spleen, they were still
above the normal range (Table). These
findings were consistent with those of histochemical analysis.
Recently, another laboratory independently came to a conclusion similar
to ours.15
We extended this study by using transplanted macrophages as a vehicle
for gene therapy of murine MPS VII. We infected macrophages cultivated
from MPS VII homozygous mutant mice (/) with an MFG-HBG retroviral vector, and transplanted these cells into nonmyeloablated MPS VII mice. The HBG activity in transduced macrophages cultivated from MPS VII mice was increased from 92 ± 62 (n = 4) to
10 265 ± 2325 (n = 4) nmol/h/mg, and was higher than that in
macrophages cultivated from normal mice (+/+) (8066 ± 1537
nmol/h/mg, n = 5). The human HBG-specific transcript was detected by
RT-PCR using human HBG-specific primers (data not shown). HBG
activities in the liver and spleen from MPS VII mice that received
genetically modified macrophages were increased at day 7 after
transplantation (28.5 ± 4.3 nmol/h/mg and 32.4 ± 7.3
nmol/h/mg, respectively). These values were almost the same as those
observed in animals that received normal macrophages. The activities
subsequently fell by 38 days (3.9 ± 0.8 nmol/h/mg in the liver
and 2.3 ± 0.8 nmol/h/mg in the spleen). However, HBG was
detectable histochemically at 38 days after transplantation (data not
shown) and pathologic improvement was significant (Figure 1C and F).
The extensive lysosomal storage in the liver and spleen was reduced.
Levels of GAGs in both the liver and spleen were reduced in mice
receiving transduced macrophages, but were still above the normal range
(Table 1).
Our observations indicate that macrophages could be alternative target
cells for gene therapy of MPS VII and other storage disorders. An
important advantage of this approach is that this procedure can be
carried out without myeloablation. The main drawbacks of this approach
were that terminally differentiated macrophages have a limited life
span, and transplanted macrophages did not migrate to the brain. We are
currently investigating approaches to overcome these limitations of our strategy.
Acknowledgments
We wish thank Dr Paul Robbins (University of Pittsburgh) for
providing the MFG vector and Dr Hiroshi Maeda (Seikagaku
Kogyo Co Ltd, Japan) for assaying GAG contents in the liver and spleen.
| |
Footnotes |
Submitted October 14, 1999; accepted February 1, 2000.
Supported by a grant from the Ministry of Human Health and
Welfare (Japan).
Reprints: Toya Ohashi, Department of Gene Therapy,
Institute of DNA Medicine, Jikei University School of Medicine, 3-25-8 Nishishinbashi, Minatoko, Tokyo 105-8461, Japan;
e-mail:tohashi{at}gd5.so-net.ne.jp.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Abstract
Introduction