Reduction of lysosomal storage in murine mucopolysaccharidosis type VII by transplantation of normal and genetically modified macrophages

Mucopolysaccharidosis type VII (MPS VII), which is also known as Sly syndrome, is a lysosomal storage disease caused by deficiency of human β-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.

Breeding pairs of (+/−) mice (B6.C-H2^bml/BY Bir-gus^mps/+) 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 

Macrophages from bone marrow cells were cultivated from normal mice (+/+) and MPS VII mice (−/−) as described.8 9Briefly, 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).

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).

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.

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.7HBG activity in the tissues was assayed as described.11 The concentration of GAGs in the liver and spleen was measured as described.12 

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).

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.

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.