Ainslie L.K. Derrick-Roberts

Skeletal response to lentiviral mediated gene therapy in a mouse model of MPS VII

Mucopolysaccharidosis VII (MPS VII) is an autosomal recessive, lysosomal storage disorder caused by β-glucuronidase (GUSB) deficiency, resulting in the accumulation of glycosaminoglycans (GAGs), in a variety of cell types. Severe, progressive skeletal pathology, termed dysostosis multiplex, is a prominent clinical feature of MPS VII. We have evaluated a gene therapy protocol for its efficacy in preventing the development and progression of bone pathology in MPS VII mice treated with a lentiviral vector at birth or at 7 weeks. Two weeks after injections, high levels of vector expression were observed in liver, spleen and bone marrow and to a lesser extent in kidney, lung and heart. Widespread clearance of GAG storage was observed in somatic tissues of both groups and some clearance of neuronal storage was observed in mice treated from birth. Micro-CT analysis demonstrated a significant decrease in vertebral and femoral bone mineral volume, trabecular number, bone surface density and cortical bone thickness in both treatment groups. Lumbar and femoral bone lengths were significantly decreased in untreated MPS VII mice, while growth plate heights were increased and these parameters did not change upon treatment. Small improvements in performance in the open field and rotarod behaviour tests were noted. Overall, systemic lentiviral-mediated gene therapy results in a measurable improvement in parameters of bone mass and architecture as well as biochemical and enzymatic correction. Conversely, growth plate chondrocytes were not responsive to treatment, as evidenced by the lack of improvement in vertebral and femoral bone length and growth plate height.

Lentiviral-mediated gene therapy results in sustained expression of β-glucuronidase for up to 12 months in the gus(mps/mps) and up to 18 months in the gus(tm(L175F)Sly) mouse models of mucopolysaccharidosis type VII.

A number of mucopolysaccharidosis type VII (MPS VII) mouse models with different levels of residual enzyme activity have been created replicating the range of clinical phenotypes observed in human MPS VII patients. In this study, a lentivirus encoding murine β-glucuronidase was administered intravenously at birth to both the severe (Gus(mps/mps) strain) and attenuated (Gus(tm(L175F)Sly) strain) mouse models of MPS VII. Circulating enzyme levels were normalized in the Gus(mps/mps) mice and were 3.5-fold higher than normal in the Gus(tm(L175F)Sly) mouse 12 and 18 months after administration. Tissue β-glucuronidase activity increased over untreated levels in all tissues evaluated in both strains at 12 months, and the elevated level was maintained in Gus(tm(L175F)Sly) tissues at 18 months. These elevated enzyme levels reduced glycosaminoglycan storage in the liver, spleen, kidney, and heart in both models. Bone mineral volume decreased toward normal in both models after 12 months of therapy and after 18 months in the Gus(tm(L175F)Sly) mouse. Open-field exploration was improved in 18-month-old treated Gus(tm(L175F)Sly) mice, while spatial learning improved in both 12- and 18-month-old treated Gus(tm(L175F)Sly) mice. Overall, neonatal administration of lentiviral gene therapy resulted in sustained enzyme expression for up to 18 months in murine models of MPS VII. Significant improvements in biochemistry and enzymology as well as functional improvement of bone and behavior deficits in the Gus(tm(L175F)Sly) model were observed. Therapy significantly increased the lifespan of Gus(mps/mps) mice, with 12 months being the longest reported lentiviral treatment for this strain. It is important to assess the long-term outcome on enzyme levels and effect on pathology for lentiviral gene therapy to be a potential therapy for MPS patients.

Correction of murine mucopolysaccharidosis type IIIA central nervous system pathology by intracerebroventricular lentiviral‐mediated gene delivery

Mucopolysaccharidoses (MPS) are inborn metabolic disorders caused by a deficiency of glycosaminoglycan degrading enzymes. Although intravenous enzyme replacement therapy is a viable approach for the treatment of non‐neuronopathic forms of MPS, its effectiveness in the central nervous system (CNS) is limited by the blood–brain barrier. Alternatively, enzyme replacement therapies and other therapies that directly target the brain represent approaches that circumvent the blood–brain barrier and, in the case of gene therapies, are intended to negate the need for repetitive dosing.

Reversal of established bone pathology in MPS VII mice following lentiviral-mediated gene therapy

Severe, progressive skeletal dysplasia is a major symptom of multiple mucopolysaccharidoses (MPS) types. While a gene therapy approach initiated at birth has been shown to prevent the development of bone pathology in different animal models of MPS, the capacity to correct developed bone disease is unknown. In this study, ex vivo micro-computed tomography was used to demonstrate that bone mass and architecture of murine MPS VII L5 vertebrae were within the normal range at 1month of age but by 2months of age were significantly different to normal. The difference between normal and MPS VII BV/TV increased with age reaching a maximal difference at approximately 4months of age. In mature MPS VII bone BV/TV is increased (51.5% versus 21.5% in normal mice) due to an increase in trabecular number (6.2permm versus 3.8permm in normal mice). The total number of osteoclasts in the metaphysis of MPS VII mice was decreased, as was the percentage of osteoclasts attached to bone. MPS VII osteoblasts produced significantly more osteoprotegerin (OPG) than normal osteoblasts and supported the production of fewer osteoclasts from spleen precursor cells than normal osteoblasts in a co-culture system. In contrast, the formation of osteoclasts from MPS VII spleen monocytes was similar to normal in vitro, when exogenous RANKL and m-CSF was added to the culture medium. Administration of murine β-glucuronidase to MPS VII mice at 4months of age, when bone disease was fully manifested, using lentiviral gene delivery resulted in a doubling of osteoclast numbers and a significant increase in attachment capacity (68% versus 29.4% in untreated MPS VII animals). Bone mineral volume rapidly decreased by 39% after gene therapy and fell within the normal range by 6months of age. Collectively, these results indicate that lentiviral-mediated gene therapy is effective in reversing established skeletal pathology in murine MPS VII.

Rhodamine B and 2-acetamido-1,3,6-tri-O-acetyl-4-deoxy-4-fluoro-d-glucopyranose (F-GlcNAc) inhibit chondroitin/dermatan and keratan sulphate synthesis by different mechanisms in bovine chondrocytes

MPS disorders result from a deficiency or absence of glycosaminoglycan (GAG) degrading enzymes leading to an imbalance between the synthesis and degradation of GAGs and their subsequent accumulation in a range of cells. The inhibition of GAG synthesis using small chemical inhibitors has been proposed as a novel therapeutic approach to treatment. Several inhibitors have been shown to decrease heparan sulphate GAG synthesis and in this study we evaluated a novel fluorinated analog of N-acetylglucosamine (2-acetamido-1,3,6-tri-O-acetyl-4-deoxy-4-fluoro-D-glucopyranose (F-GlcNAc)) and rhodamine B for their ability to also inhibit the synthesis of chondroitin/dermatan and keratan sulphate GAGs present in bovine cartilage. Both inhibitors decreased GAG synthesis in chondrocyte monolayer culture and in cartilage chip explant culture in a dose dependent manner. Both inhibitors decreased the size of newly synthesised proteoglycans and in the case of F-GlcNAc this was due to a decrease in newly synthesised GAG chain size. Rhodamine B, however, did not affect GAG chain size, while both inhibitors decreased the amount of chondroitin/dermatan and keratan sulphate GAG equally. The expression of genes responsible for the initiation and elongation of chondroitin/dermatan sulphate and keratan sulphate GAGs were downregulated in the presence of rhodamine B but not in the presence of F-GlcNAc. Thus the 2 inhibitors appear to have differing effects on GAG synthesis, with F-GlcNAc inhibiting the epimerisation of UDP-GlcNAc to UDP-GalNAc thus decreasing the availability of monosaccharides for addition to the growing GAG chain, whereas rhodamine B is more likely to reduce the number of GAG chains. Together with previous data these 2 inhibitors are capable of non-specific inhibition of GAG synthesis, reducing the production of chondroitin/dermatan sulphate, keratan sulphate and heparan sulphate GAGs. As such they would be applicable to therapy in a range of MPS disorders.

Substrate Deprivation Therapy to Reduce Glycosaminoglycan Synthesis Improves Aspects of Neurological and Skeletal Pathology in MPS I Mice

Mucopolysaccharidosis type I (MPS I) is the most common form of the MPS group of genetic diseases. MPS I results from a deficiency in the lysosomal enzyme α-l-iduronidase, leading to accumulation of undegraded heparan and dermatan sulphate glycosaminoglycan (GAG) chains in patient cells. MPS children suffer from multiple organ failure and die in their teens to early twenties. In particular, MPS I children also suffer from profound mental retardation and skeletal disease that restricts growth and movement. Neither brain nor skeletal disease is adequately treated by current therapy approaches. To overcome these barriers to effective therapy we have developed and tested a treatment called substrate deprivation therapy (SDT). MPS I knockout mice were treated with weekly intravenous injections of 1 mg/kg rhodamine B for six months to assess the efficacy of SDT. Mice were assessed using biochemistry, micro-CT and a battery of behaviour tests to determine the outcome of treatment. A reduction in female bodyweight gain was observed with the treatment as well as a decrease in lung GAG. Behavioural studies showed slight improvements in inverted grid and significant improvements in learning ability for female MPS I mice treated with rhodamine B. Skeletal disease also improved with a reduction in bone mineral volume observed. Overall, rhodamine B is safe to administer to MPS I knockout mice where it had an effect on improving aspects of neurological and skeletal disease symptoms and may therefore provide a potential therapy or adjunct therapy for MPS I patients.

N-butyldeoxynojirimycin treatment restores the innate fear response and improves learning in mucopolysaccharidosis IIIA mice

UNLABELLED Mucopolysaccharidosis IIIA is a heritable neurodegenerative disorder resulting from the dysfunction of the lysosomal hydrolase sulphamidase. This leads to the primary accumulation of the complex carbohydrate heparan sulphate in a wide range of tissues and the secondary neuronal storage of gangliosides GM2 and GM3 in the brain. GM2 storage is associated with CNS deterioration in the GM2 gangliosidosis group of lysosomal storage disorders and may also contribute to MPS CNS disease. N-butyldeoxynojirimycin, an inhibitor of ceramide glucosyltransferase activity and therefore of ganglioside synthesis, was administered to MPS IIIA mice both prior to maximal GM2 and GM3 accumulation (early treatment) and after the maximum level of ganglioside had accumulated in the brain (late treatment) to determine if behaviour was altered by ganglioside level. Ceramide glucosyltransferase activity was decreased in both treatment groups; however, brain ganglioside levels were only decreased in the late treatment group. Learning in the water cross maze was improved in both groups and the innate fear response was also restored in both groups. A reduction in the expression of inflammatory gene Ccl3 was observed in the early treatment group, while IL1β expression was reduced in both treatment groups. Thus, it appears that NB-DNJ elicits a transient decrease in brain ganglioside levels, some modulation of inflammatory cytokines and a functional improvement in behaviour that can be elicited both before and after overt neurological changes manifest. SYNOPSIS NB-DNJ improves learning and restores the innate fear response in MPS IIIA mice by decreasing ceramide glucosyltransferase activity and transiently reducing ganglioside storage and/or modulating inflammatory signals.

Delayed development of ossification centers in the tibia of prenatal and early postnatal MPS VII mice

Short stature is a characteristic feature of most of the mucopolysaccharidoses, a group of inherited lysosomal storage disorders caused by a single enzyme deficiency. MPS patients present with progressive skeletal defects from an early age, including short stature due to impaired cartilage-to-bone conversion (endochondral ossification). The aim of this study was to determine which murine MPS model best reproduces the bone length reduction phenotype of human MPS and use this model to determine the earliest developmental stage when disrupted endochondral ossification first appears. Gusmps/mps mice representing severe MPS VII displayed the greatest reduction in bone elongation and were chosen for histopathological analysis. Tibial development was assessed from E12.5 to 6 months of age. Chondrocytes in the region of the future primary ossification center became hypertrophic at a similar age to normal in the MPS VII mouse fetus, but a delay in bone deposition was observed with an approximate 1 day delay in the formation of the primary ossification centre. Likewise, chondrocytes in the region of the future secondary ossification center also became hypertrophic at the same age as normal in the MPS VII early postnatal mouse. Bone deposition in the secondary ossification centre was delayed by two days in the MPS VII proximal tibia (observed at postnatal day 14 (P14) compared to P12 in normal). The thickness of the tibial growth plate was larger in MPS VII mice from P9 onwards. Abnormal endochondral ossification starts in utero in MPS VII and worsens with age. It is characterized by a normal timeframe for chondrocyte hypertrophy but a delay in the subsequent deposition of bone in both the primary and secondary ossification centres, accompanied by an increase in growth plate thickness. This suggests that the signals for vascular invasion and bone deposition, some of which are derived from hypertrophic chondrocytes, are altered in MPS VII.