Radiographic evaluation of bones and joints in mucopolysaccharidosis I and VII dogs after neonatal gene therapy

Abstract

Mucopolysaccharidosis I (MPS I) and MPS VII are due to deficient activity of the glycosaminoglycan-degrading lysosomal enzymes alpha-L-iduronidase and beta-glucuronidase, respectively, and result in abnormal bones and joints. Here, the severity of skeletal disease in MPS I and MPS VII dogs and the effects of neonatal gene therapy were evaluated. For untreated MPS VII dogs, the lengths of the second cervical vertebrae (C2) and the femur were only 56% and 84% of normal, respectively, and bone dysplasia and articular erosions, and joint subluxation were severe. Previously, we reported that neonatal intravenous injection of a retroviral vector (RV) with the appropriate gene resulted in expression in liver and blood cells, and high serum enzyme activity. In this study, we demonstrate that C2 and femurs of RV-treated MPS VII dogs were longer at 82% and 101% of normal, respectively, and there were partial improvements of qualitative abnormalities. For untreated MPS I dogs, the lengths of C2 and femurs (91% and 96% of normal, respectively) were not significantly different from normal dogs. Qualitative changes in MPS I bones and joints were generally modest and were partially improved with RV treatment, although cervical spine disease was severe and was difficult to correct with gene therapy in both models. The greater severity of skeletal disease in MPS VII than in MPS I dogs may reflect accumulation of chondroitin sulfate in cartilage in MPS VII, or could relate to the specific mutations. Neonatal RV-mediated gene therapy ameliorates, but does not prevent, skeletal disease in MPS I and MPS VII dogs.

Figure 1. Radiographs of vertebrae

Radiographs from male dogs were obtained at the ages in years (Y) shown in the lower left corner. The genotype and treatment status are indicated above the panels. RV-treated dogs received neonatal IV injection of an RV expressing the appropriate gene. Examples of RV-treated MPS I dogs are from I-171 in panels C and H and I-172 in panel M, while all examples of MPS VII dogs are from M1328. The scale bar shown in each image is 1 cm, and the cranial aspect is at the top and the caudal aspect at the bottom. A–E. Ventrodorsal view of the cervical spine. The second cervical vertebra (C2), C3, and C4 are indicated. The horizontal white arrows indicate intervertebral spaces. Black vertical arrows indicate the medial and lateral borders of the pedicle, and the space between the arrows was used to assess width. F–J. Lateral view of the cervical spine. Slanted white arrows indicate caudoventral vertebral body beaking. The black horizontal arrow indicates fusion of the articular facet joint. K–O. Lateral lumbar spine. The sixth lumbar vertebra (L6) and L7 are indicated. Horizontal white arrows indicate intervertebral spaces.

Figure 2. Axial skeleton measurements

Measurements of vertebral body and intervertebral space lengths were obtained for the cervical and lumbar spine in post-pubertal dogs at 0.7 to 3 years after birth, and values for males and females were combined. Some RV-treated MPS VII dogs were evaluated at several ages, and values (which did not vary substantially) were averaged to give a single value for that animal. Values in several animals were then averaged and the mean +/− the standard deviation was calculated. The means for several animals in each group +/− one SD are shown; * indicates p of 0.01 to 0.05 and ** indicates p<0.01 for comparison of the groups connected by a bracket using ANOVA with Tukey post-hoc analysis. A. Vertebral body lengths for MPS I dogs. Vertebral body lengths of C2 (second cervical vertebra), C3, C4, L5 (fifth lumbar vertebra), L6, and L7 were measured for 8 heterozygous normal (1 male, 7 female), 7 untreated MPS I (5 male, 2 female), and 6 RV-treated MPS I (3 male, 3 female) post-pubertal dogs at 0.6 to 1 year of age. B. Vertebral body lengths for MPS VII dogs. Lengths were measured for 12 heterozygous normal (3 male, 9 female), 15 untreated MPS VII (11 male, 4 female), and 7 RV-treated MPS VII (4 male, 3 female) dogs. C and D. Intervertebral space lengths for MPS I and VII dogs. Intervertebral space lengths were measured for the same animals whose values are shown in panels A and B.

Figure 3. Axial skeleton radiographic scores

Radiographs were scored for abnormalities at 1 year of age for most groups, where 0 indicates normal and 2 indicates severely abnormal. Both males and females were evaluated, means +/− SD are shown, and * indicates p<0.05 and ** indicates p<0.01 for comparison of the groups connected by a bracket using the Mann-Whitney U test. A. Radiographic scores for MPS I dogs. The scores are shown for 8 normal dogs at 1 to 2 years (2 male, 6 female), 6 untreated MPS I dogs at 1 year (4 male, 2 female), and 6 neonatal RV-treated MPS I dogs at 1 year (3 male, 3 female). Vertebral body dysplasia, pedicle widening, caudoventral vertebral body beaking, articular facet joint fusion, and vertebral body tipping were evaluated. B. Radiographic scores for MPS VII dogs. The scores are shown for 8 normal dogs at 1 to 2 years (2 male, 6 female), 6 untreated MPS VII dogs at 1 year (5 male, 1 female), and 5 RV-treated dogs at 1 year (2 male, 3 female), and statistical comparisons were performed for the 3 groups with the Mann-Whitney U test. Two of the seven RV-treated MPS VII dogs that were alive at 1 year were not evaluated as radiographs were not obtained at the appropriate age. RV-treated MPS VII dogs were also scored for 5 dogs at 4 to 7 years (2 males and 2 females at 7 years and 1 female at 4 years), and comparison of values with those in RV-treated MPS VII dogs at 1 year using the Mann-Whitney U test failed to find significant differences.

Figure 4. Evaluation of joints

Radiographs are from male dogs at the ages in years (Y) shown in the lower left corner. The genotype and treatment status are indicated above the panels. Examples of radiographs of RV-treated MPS I dogs were from I-172 in panels C and H, and from I-171 in panel M. All examples of radiographs of the RV-treated MPS VII dog were from M1328. A–E. Carpal joint dorsopalmar. The metacarpals are at the bottom of the image, and the radius (right) and ulna (left) are on the top. The white vertical arrows indicate radiocarpal bones. The slanted white arrow indicates erosions of the 3^rd metacarpal bone. F–J. Coxofemoral joint ventrodorsal. The pelvis is on the left side of the image. The horizontal white arrow indicates luxation of the femoral head. K–O. Stifle joint lateral. The femur is at the top of the image and the tibia is at the bottom. The white arrows indicate the left aspect of the triangular-shaped radiolucent fat pad present at the cranial aspect of the stifle joint, which can be visualized when no effusion is present. This triangle is absent in an untreated MPS VII dog (panel N) due to an effusion.

Figure 5. Appendicular skeleton radiographic scores. A. Radiographic scores for MPS I carpi

Radiographic scores are shown for 8 normal dogs at 2 years of age (2 male, 6 female), 6 untreated MPS I dogs at 1 year (4 male, 2 female), and 6 RV-treated MPS I dogs at 1 year of age (3 male, 3 female). Carpal bone dysplasia, carpal bone lucency, articular bone erosions, carpal joint subluxation, and joint effusions were scored from 0 (normal or absent) to 2 (severely abnormal). B. Radiographic scores for MPS VII forelimb. Radiographic scores are shown for 8 normal dogs at 1 to 2 years of age (2 male, 6 female), 6 untreated MPS VII dogs at 1 year (5 male, 1 female), 5 RV-treated dogs at 1 year (2 males and 2 females at 7 years, and 1 female at 4 years), and 5 RV-treated dogs at >4 years (2 male, 3 female). C. Radiographic scores for MPS I hindlimb. Radiographic scores are shown for the same groups that are described in panel A. The coxofemoral joint was evaluated for dysplasia, subluxation, articular bone erosions, and degenerative joint disease (DJD). The stifle joints were evaluated for patellar luxation (pat. lux.) and effusions. D. Radiographic scores for MPS VII hindlimb. Radiographic scores are shown for the same animals described in panel B. E. Radiographic scores for MPS I femur and radius/ulna. Radiographic scores are shown for the same dogs described in panel A. Femoral head dysplasia, femoral erosions, femoral curvature, radius/ulna curving, and radius/ulna dysplasia were evaluated. F. Radiographic scores for MPS VII femur and radius/ulna. Radiographic scores are shown for the same dogs described in panel B.

Figure 6. Appendicular skeleton lengths

Measurements of bone length were obtained for hindlimbs for post-pubertal dogs that were 0.7 to 3 years of age. Values did not differ in the RV-treated animals that were radiographed at 1 to 3 years of age. * indicates a p value of 0.01 to 0.05, and ** indicates a p value <0.01 for the statistical comparison of the groups that are connected with a bracket. A. Hindlimb bone lengths in MPS I dogs. Measurements of the femur, tibia, and metatarsals (MT) of the same dogs that are described in Fig. 5A are shown. B. Hindlimb bone lengths in MPS VII dogs. Measurements of the bones of the dogs described in Fig. 5B are shown.