Dose-dependent effects of enzyme replacement therapy on skeletal disease progression in mucopolysaccharidosis VII dogs

Abstract

Mucopolysaccharidosis (MPS) VII is an inherited lysosomal storage disorder characterized by deficient activity of the enzyme β-glucuronidase. Skeletal abnormalities are common in patients and result in diminished quality of life. Enzyme replacement therapy (ERT) for MPS VII using recombinant human β-glucuronidase (vestronidase alfa) was recently approved for use in patients; however, to date there have been no studies evaluating therapeutic efficacy in a large animal model of MPS VII. The objective of this study was to establish the effects of intravenous ERT, administered at either the standard clinical dose (4 mg/kg) or a high dose (20 mg/kg), on skeletal disease progression in MPS VII using the naturally occurring canine model. Untreated MPS VII animals exhibited progressive synovial joint and vertebral bone disease and were no longer ambulatory by age 6 months. Standard-dose ERT-treated animals exhibited modest attenuation of joint disease, but by age 6 months were no longer ambulatory. High-dose ERT-treated animals exhibited marked attenuation of joint disease, and all were still ambulatory by age 6 months. Vertebral bone disease was recalcitrant to ERT irrespective of dose. Overall, our findings indicate that ERT administered at higher doses results in significantly improved skeletal disease outcomes in MPS VII dogs.

Keywords: Sly syndrome; bone; canine; enzyme replacement therapy; lysosomal storage disorder; mucopolysaccharidosis; spine; synovial joint; vestronidase alfa.

Graphical abstract

Figure 1

Tissue enzyme distribution and glycosaminoglycan content for skeletal tissues and visceral organs (A) β-Glucuronidase (GUSB) activity, (B) β-hexosaminidase (HEX) activity, and (C) glycosaminoglycan (GAG) content. Results are normalized to total protein content. ∗p < 0.05 versus Control; +p < 0.05 versus MPS VII Untreated; n = 3–6 per group; Kruskal-Wallis tests with post-hoc Dunn’s tests. Data are presented as median and interquartile range.

Figure 2

Plain radiographs of the lower appendicular skeleton (A) Ventrodorsal and (B) lateral views at age 3 and 6 months. The blue arrows indicate coxofemoral subluxation, the yellow arrows indicate genu valgum and the red arrow indicates patellar dislocation. Joint abnormalities exhibited dose-dependent improvements with ERT.

Figure 3

Magnetic resonance imaging of stifle joints (A) Representative sagittal T2-weighted, fat saturated and (B) dorsal (coronal) proton density-weighted images illustrating pathological features of synovial joint disease in MPS VII dogs, including effusion synovitis cranial to the joint (red asterisks), Baker’s cysts caudal to the joint (blue asterisks), patellar displacement (orange arrows), and meniscal extrusion (green arrow). Joint abnormalities exhibited dose-dependent improvements with ERT. F, femur (distal); T, tibia (proximal); P, patella. (C–L) Semi-quantitative grading of pathological features assessed from MRI images. Overall grade is the sum of individual scores. ∗p < 0.05 versus Control; +p < 0.05 versus MPS VII Untreated; n = 3–5 per group; Kruskal-Wallis tests with post-hoc Dunn’s tests. Data are presented as median and interquartile range.

Figure 4

Histological evaluation of stifle joint articular cartilage (A) Representative mid-sagittal sections of the medial femoral condyle. Scale bars, 2 mm (safranin-O and fast green stain). (B) Higher-magnification views of the regions indicated by the box in (A). Scale bar, 100 μm. (C–F) Semi-quantitative grading of cartilage pathology. Overall grade is the sum of individual grades. ∗p < 0.05 versus Control; n = 3–5 per group; Kruskal-Wallis tests with post-hoc Dunn’s tests. Data are presented as median and interquartile range.

Figure 5

Expression levels of inflammatory mediators in synovial fluid ∗p < 0.05 versus Control; +p < 0.05 versus MPS VII Untreated; ∼p < 0.05 versus MPS VII ERT SD; n = 3–6 per group; Kruskal-Wallis tests with post-hoc Dunn’s tests. Data are presented as median and interquartile range.

Figure 6

Biomechanical properties of CCLs (A) Representative image of CCL undergoing uniaxial tensile testing. T, tibia (proximal); F, femur (distal); arrows, testing direction. (B) Stiffness, (C) modulus, (D) toughness, (E) failure load, (F) failure stress, (G) failure strain. ∗p < 0.05 versus Control; n = 3–6 per group; Kruskal-Wallis tests with post-hoc Dunn’s tests. Data are presented as median and interquartile range.

Figure 7

Plain radiographs and microCT imaging of vertebral bodies, and serum BAP activity (A) Representative radiographs of lumbar spine vertebrae at age 3 and 6 months. Arrows indicate radiolucent lesions at the ventral margins, which were present in all MPS VII vertebrae regardless of treatment status. (B) Representative microCT images illustrating diminished longitudinal growth of MPS VII vertebral bodies at age 6 months regardless of treatment status. Scale bar, 3 mm. (C) Bone volume fraction (BV/TV), (D) bone mineral density (BMD), (E) trabecular thickness (Tb.Th), (F) trabecular spacing (Tb.Sp), (G) trabecular number (Tb.N), (H) connectivity density (Conn.Dens), (I) Serum BAP. ∗p < 0.05 versus Control; +p < 0.05 versus MPS VII Untreated; n = 3–6 per group; Kruskal-Wallis tests with post-hoc Dunn’s tests. Data are presented as median and interquartile range.

Figure 8

Histological evaluation of vertebral bodies (A) Representative mid-sagittal sections of T13 vertebra illustrating diminished longitudinal growth and bone content in primary ossification centers (poc) of MPS VII vertebrae regardless of treatment status. Scale bar, 2 mm (Alcian blue and picrosirius red stain). (B) Higher-magnification images of the regions indicated by boxes in (A), illustrating the presence of abnormal cartilage in the secondary ossification centers (soc) of MPS VII vertebrae, regardless of treatment status. Scale bar, 500 μm. (C) Representative mid-sagittal sections of vertebral growth plates illustrating diminished cellularity in MPS VII animals regardless of treatment status. Scale bar, 100 μm (hematoxylin and eosin stain). (D–G) Quantification of growth plate proliferative and hypertrophic zone (pz and hz, respectively) height and cellularity. ∗p < 0.05 versus Control; n = 3–6 per group; Kruskal-Wallis tests with post-hoc Dunn’s tests. Data are presented as median and interquartile range.