Effects of enzyme replacement therapy started late in a murine model of mucopolysaccharidosis type I

Abstract

Mucopolysaccharidosis type I (MPS I) is a progressive disorder caused by deficiency of α-L-iduronidase (IDUA), which leads to storage of heparan and dermatan sulphate. It is suggested that early enzyme replacement therapy (ERT) leads to better outcomes, although many patients are diagnosed late and don't receive immediate treatment. This study aims to evaluate the effects of late onset ERT in a MPS I murine model. MPS I mice received treatment from 6 to 8 months of age (ERT 6-8mo) with 1.2mg laronidase/kg every 2 weeks and were compared to 8 months-old wild-type (Normal) and untreated animals (MPS I). ERT was effective in reducing urinary and visceral GAG to normal levels. Heart GAG levels and left ventricular (LV) shortening fraction were normalized but cardiac function was not completely improved. While no significant improvements were found on aortic wall width, treatment was able to significantly reduce heart valves thickening. High variability was found in behavior tests, with treated animals presenting intermediate results between normal and affected mice, without correlation with cerebral cortex GAG levels. Cathepsin D activity in cerebral cortex also did not correlate with behavior heterogeneity. All treated animals developed anti-laronidase antibodies but no correlation was found with any parameters analyzed. However, intermediary results from locomotion parameters analyzed are in accordance with intermediary levels of heart function, cathepsin D, activated glia and reduction of TNF-α expression in the cerebral cortex. In conclusion, even if started late, ERT can have beneficial effects on many aspects of the disease and should be considered whenever possible.

Fig 1. Urinary and tissue GAG levels.

A) MPS I mice treated from 6 to 8 months with 1.2mg laronidase/kg every two weeks (n = 10). Urine was collected 1 day post injections. **P<0.01, ANOVA and Tukey. B) Comparison between urinary GAG from 8-month old wild-type (normal, n = 4), MPS I (n = 3) and treated MPS I mice (ERT 6–8mo, n = 10), 1 day after last injection. Treated mice achieve normal GAG level (dotted lines) as soon as the second injection. C) Tissue GAG from 8-month old normal (n = 4/5), MPS I (n = 5–6) and ERT 6–8mo (n = 10) mice collected two weeks after last injection. #P<0.05, Kruskal-Wallis and Mann-Whitney post hoc. ***P<0.001 ANOVA and Tukey post hoc.

Fig 2. Heart function.

A) GAG storage in hearts from 8-month old normal (n = 3), MPS I (n = 5) and MPS I mice treated with ERT from 6 to 8 months (ERT 6–8mo, n = 10), 2 weeks after last injection. B-D: Echocardiography results from 8-month old normal (n = 6/8), MPS I (n = 6/10) and MPS I mice treated with ERT from 6 to 8 months (ERT 6–8mo, n = 10), 1 day after last injection. B) Ejection fraction, C) Shortening fraction D) Ratio between ejection and acceleration times measured at the pulmonary valve. ***P≤0.001, **P≤0.006 ANOVA and Tukey post hoc.

Fig 3. Heart valves and Aorta.

A) Heart valve thickness and B) Aortic wall width in 8-month old normal (n = 3/4), MPS I (n = 3/5) and 6 to 8 months laronidase treated MPS I (ERT 6–8mo, n = 5/10), 2 weeks after last injection. *P≤0.03, ANOVA and Tukey post hoc. C) Heart valves and aortas stained with H-E and Alcian Blue. The presence of large vacuoles and intense alcian blue staining indicating GAG storage are evident in thick MPS I mice valves but are significantly reduced in treated animals. A large amount of vacuoles are also seen in the dilated MPS I and ERT 6–8mo aortas. Original magnification, ×400.

Fig 4. Open field test.

Results from 8-month old normal (n = 7), MPS I (n = 11) and 6 to 8 months laronidase treated MPS I (ERT 6–8mo, n = 10), 2 weeks after last injection. #P≤0.04, Kruskal-Wallis and Mann-Whitney post hoc.

Fig 5. Brain disease markers.

A) Cerebral cortex GAG levels and B) Cathepsin D activity in 8-month old normal (n = 5), MPS I (n = 5) and 6 to 8 months laronidase treated MPS I (ERT 6–8mo, n = 10), 2 weeks after last injection. *P = 0.02, ANOVA and Tukey. ###P<0.001, Kruskal-Wallis and Mann-Whitney post hoc.

Fig 6. Neuroinflammatory markers.

A-C) Representative sections from 8-month normal (A, n = 5), MPS I (B, n = 5) and 6 to 8 months MPS I laronidase treated (C, n = 10) mouse cerebral cortex stained for glial fibrilary acidic protein (GFAP). D) Number of positive cells counted in 5 fields (400× magnification) in mouse cortex at 8 months. *P ≤0.02. ANOVA and Tukey. E) MIP1-α and TNF-α mRNA expression on cerebral cortex normalized by GAPDH expression. ###P≤0.001, #P ≤0.02 Kruskal-Wallis and Mann-Whitney post hoc.