Neonatal retroviral vector-mediated hepatic gene therapy reduces bone, joint, and cartilage disease in mucopolysaccharidosis VII mice and dogs
Introduction
The mucopolysaccharidoses (MPSs) are lysosomal storage diseases in which glycosaminoglycans (GAGs) accumulate in lysosomes due to deficiencies in enzymes involved in their catabolism [1]. Clinical manifestations of most MPS syndromes include hepatosplenomegaly, corneal clouding, reduced hearing, neurological dysfunction, and cardiovascular disease. In addition, bone, joint, and cartilage disease is a serious problem in MPS I, II, IV, VI, and VII, which involve enzymes that degrade chondroitin sulfate or dermatan sulfate. In these disorders, affected individuals have short and thick limbs, a flattened face, swollen joints that limit movement, and narrowing of the internal diameter of the trachea that can contribute to upper airway disease and lead to difficulties in intubation for anaesthesia [1], [2]. The accumulation of GAGs in connective tissues likely contributes to these manifestations. GAGs are polymers of sugars that are synthesized on proteins referred to as proteoglycans (PG). PG are components of extracellular matrices, are involved in cell:cell interactions, and can augment the effect of several growth factors [3], [4].
The intracellular accumulation of GAGs in MPS may lead to dysfunction of cells involved in bone and cartilage formation and remodeling [5]. Lysosomes in osteocytes and osteoblasts, and chondrocytes of the growth plate, accumulate storage, which may contribute to the reduced in vivo bone formation reported in MPS VII mice after calcein labeling [6] and to reduced bone lengths reported for MPS VII mice [5]. Osteoclasts from MPS VII animals have been reported to fail to adhere tightly to bone in vivo and to have reduced activity in vitro [6], which might impair bone remodeling and contribute to bone sclerosis. Chondrocytes of the articular cartilage also have large amounts of lysosomal storage [5], [7], which may be responsible for the increased apoptosis of articular chondrocytes in MPS VI rats and cats [8] and degenerative joint disease. Synoviocytes contain lysosomal storage, which may result in hyperplasia of the synovium and development of joint effusions. It is also possible that GAGs are released from diseased cells, and these extracellular GAGs inhibit the cell signaling or adhesion properties of PG, affect collagen biosynthesis, or have other adverse effects. Indeed, chondroitin-4 sulfate inhibits matrix mineralization in vitro [9], [10], and dermatan sulfate induces secretion of inflammatory molecules in cultured chondrocytes from normal animals [8].
MPS VII is due to deficient β-glucuronidase (GUSB; EC 3.2.1.31) activity. GUSB contributes to the degradation of chondroitin-4 and -6 sulfates, dermatan sulfate, and heparan sulfate [1]. Although rare, MPS VII provides a convenient model of MPS due to the availability of mice [11], dogs [12], and cats [13] with the disease, and a histochemical stain for GUSB activity. Some progress has been made in alleviating the bone disease in MPS VII mice with enzyme replacement therapy (ERT), bone marrow transplantation (BMT), or liver-directed gene therapy. ERT involves IV injection of GUSB with mannose 6-phosphate (M6P), which can be transported to the lysosome by the M6P receptor [14]. Although initiation of ERT in newborns reduces bone disease in MPS VII mice [15], [16], [17], the expense and the need for frequent infusions will restrict its use. BMT can reduce bone disease in MPS VII mice if performed early in life [18], [19], which is likely due to localized cross-correction by BM or BM-derived cells. However, morbidity and the requirement for a compatible donor limit BMT. Liver-directed gene therapy is an attractive treatment option, as it results in the continuous secretion of GUSB with M6P by hepatocytes. Others have previously reported that neonatal AAV vector-mediated [20], [21], [22] or adenoviral vector-mediated [23], [24] gene therapy can improve some, but not all, aspects of bone disease in mice.
We previously reported that intravenous (IV) injection of neonatal mice with a retroviral vector (RV) expressing canine GUSB (cGUSB) resulted in stable expression of GUSB at levels that were >10-fold higher [25] than those reported in other studies in which bone pathology was evaluated after liver-directed gene therapy. We have also reported previously that MPS VII dogs treated with a similar neonatal gene therapy approach achieved stable levels of GUSB in serum, and had improved mobility and radiographic evidence of reduced skeletal disease [26]. Here we report further the effect of this treatment on bone, cartilage, and synovial disease in MPS VII mice and dogs.
Section snippets
Animal procedures
MPS VII mice and homozygous normal controls in a B6.C-H-2bm1/ByBir background [11] were maintained in a pathogen-free environment on an ad libitum diet with 11% fat (PicoLab Mouse Chow 20 5058). Dogs were raised in the animal colony of the School of Veterinary Medicine, University of Pennsylvania, under NIH and USDA guidelines for the care and use of animals in research. They were housed at 21 °C with ad libitum food and water and 12–15 air changes per hour. MPS VII animals were injected IV with
Mouse facial morphology and body weight
MPS VII mice were injected IV at 2–3 days after birth with a Moloney murine leukemia-based RV vector expressing the cGUSB cDNA. GUSB activity in the serum was maintained at stable levels ranging from 581 to 10,070 U/ml in individual mice for 6 months, as previously reported [25] and summarized in Table 1. Fig. 1A shows that an untreated MPS VII mouse had a short, broad face at 6 months after birth. In contrast, the facial morphology of an RV-treated mouse with high serum GUSB activity was almost
Discussion
Bone, joint, and cartilage disease results in significant clinical and cosmetic problems in patients with MPS VII that might be treated with liver-directed gene therapy. There are two potential mechanisms by which this neonatal gene approach might improve bone, joint, and cartilage disease. First, the mannose 6-phosphorylated enzyme that is secreted into blood from the transduced liver [25] could get taken up by cells in other organs via the M6PR, in a process that would resemble ERT.
Acknowledgements
This work was supported by the National Institutes of Health (R01 DK54061 and K02 DK0275 awarded to K.P.P.; DK57586 awarded to M.S.S.; DK54481 and RR02512 awarded to M.E.H.; and the pathology core of the Washington University Digestive Diseases Research Core Center Grant P30 DK52574). Robert L. Mango was supported by a Howard Hughes Medical Institute undergraduate fellowship. We thank Marie Roberts for assistance with mouse care, and Patty O'Donnell and Jean Zweigle for assistance with dog care.
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2015, Molecular Genetics and MetabolismCitation Excerpt :Gusb−/− mice that were treated neonatally with an IV injection of an RV expressing canine Gusb were evaluated to determine the effects on bone disease. Bone lengths were partially improved relative to untreated Gusb−/− mice, which was consistent with our previous results in mice treated with the same vector that had similar levels of expression [10] and with our results in RV-treated Gusb−/− dogs [8,9]. In contrast, manifestations of DJD such as dysplasia and bone irregularities were not prevented in the RV-treated mice.
The effect of neonatal gene therapy on skeletal manifestations in mucopolysaccharidosis VII dogs after a decade
2013, Molecular Genetics and MetabolismCitation Excerpt :One gene therapy approach for MPS involves the intravenous (IV) injection of a gamma retroviral vector (RV) expressing the appropriate enzyme into newborn animals, which leads to transduction of liver cells, secretion of M6P-modified enzyme into blood, and uptake of enzyme by cells in other organs via the M6P receptor [41–47]. We have previously demonstrated in MPS VII dogs that neonatal IV injection of an RV expressing β-glucuronidase (GUSB) improved long bone lengths of adults [42,43], reduced erosions of femoral heads at 6 months of age [44,47], improved mobility at 1 year [42], and reduced radiographic evidence of degenerative changes in some joints at 7 years [43]. However, it failed to prevent spine disease at 6 to 11 years of age [45].