Recombinant human alpha-N-acetylglucosamine-6-sulfatase delivered to Sanfilippo D mice with repeated intracerebroventricular injections corrects CNS pathology

Abstract

Mucopolysaccharidosis type IIID (MPS IIID; Sanfilippo D) is caused by biallelic pathogenic variants in N-acetylglucosamine-6-sulfatase (GNS), which participates in catabolism of heparan sulfate (HS) glycosaminoglycans. Characterization of MPS IIID disease at a cellular level has not been robustly achieved. We used unbiased quantitative proteomics to establish a cellular phenotype for MPS IIID mice. Recombinant human GNS (rhGNS), a variant of which previously demonstrated single dose efficacy in MPS IIID human fibroblasts and in MPS IIID neonatal mice, was used to establish a repeat dosing schedule to treat MPS IIID mice. Adult Gns KO mice or heterozygous carriers were treated via intracerebroventricular (ICV) injections and received 3, 30, or 200 μg rhGNS in 4 doses over 2 weeks or vehicle. Twenty-four hours after the final dose, HS in brain and CSF showed dose-dependent reductions, reaching carrier levels in the higher dose groups. Furthermore, the proteomic perturbations that we described were corrected by rhGNS treatment. Next, Gns KO or carrier adult mice were treated via ICV and received 3, 30 or 200 μg rhGNS or vehicle once every two weeks (Day 1, 15, 29, 43, 57, 71, 85) and were euthanized on day 91. Following treatment, total HS and MPS IIID-specific HS (GlcNAc6S) showed dose-dependent reductions in brain and CSF and markers of neuroinflammation were substantially reduced. ICV enzyme replacement therapy with rhGNS restores CNS pathology of adult MPS IIID mice even with treatment at 14-day intervals, demonstrating preclinical efficacy for MPS IIID.

Fig 1. Gene Ontology Analysis of MPS IIID Mice Compared to Carrier Mice.

A) KEGG terms that are significantly different based on the 56 differentially expressed proteins between the MPS IIID mice and carrier mice. B) GO Cellular Component terms that are significantly different based on the 56 differentially expressed proteins between the MPS IIID mice and carrier mice. C) GO Biological Process terms that are significantly different based on the 56 differentially expressed proteins between the MPS IIID mice and carrier mice. D) GO Molecular Function terms that are significantly different based on the 56 differentially expressed proteins between the MPS IIID mice and carrier mice.

Fig 2. Biochemical Analysis of Mice Treated with rhGNS for 2 Weeks.

A) Diagram of a mouse brain sectioned into 3 sections from rostral to caudal with treatment injection site marked. Brains were harvested 6 days after the final rhGNS dose. Diagram modified from [6]. B) Western blots showing GNS protein levels in brains (section 2) and livers of mice treated with increasing doses of rhGNS for 4 doses over 2 weeks. GAPDH levels shown as a loading control. C) GNS activity in brains (sectioned into 3 sections from rostral to caudal) and liver. D) β-HEX activity in brains (sectioned into 3 sections from rostral to caudal) and liver. E) HS levels in the brain of mice from section 1. F) HS levels in the CSF of mice. In C-F, blue data points are male and red data points are female. P values: * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001.

Fig 3. Proteomic Analysis of Mice Treated with rhGNS for 2 Weeks.

A) Volcano plot of protein expression of MPS IIID mice treated with rhGNS relative to carrier mice. B) Volcano plot of protein expression of MPS IIID mice relative to carrier mice. C) Volcano plot of protein expression of MPS IIID mice relative to MPS IIID mice treated with rhGNS. D) PCA plot of the individual mice from all groups showing the distribution of the mice due to the relative abundances of the 56 proteins that are differentially expressed between the MPS IIID mice and the carrier mice. E) Heatmap showing the differential expression of the 56 proteins that are differentially expressed between the MPS IIID mice (treated and untreated) and the carrier mice. In D and E, “Het” labels are short for heterozygous and have the same meaning as carrier as presented elsewhere in this manuscript.

Fig 4. Immunofluorescence Analysis of Mice Treated with rhGNS for 12 Weeks.

A) representative CD68 and GFAP IF images from the somatosensory cortex and striatum from brains from each treatment group. Images are at 20X magnification and scale bar represents 100 μm. B) Quantification of IF signal from somatosensory cortex CD68 data. C) Quantification of IF signal from striatum CD68 data. D) Quantification of IF signal from somatosensory cortex GFAP data. E) Quantification of IF signal from striatum GFAP data. In the quantification data, blue data points are male and red data points are female. P values: ** < 0.01, *** < 0.001, **** < 0.0001.

Fig 5. Heparan Sulfate Levels in Mice CNS after 12 Week Treatment with rhGNS.

A) Heparan Sulfate (HS) levels in the brain of mice treated with different doses of rhGNS for 7 doses over 12 weeks. Mice were euthanized 6 days after their final dose. B) HS levels in the CSF of mice treated with increasing doses of rhGNS for 7 doses over 12 weeks. C) GlcNAc6S levels in the brain of mice. Blue data points are male and red data points are female. P values: * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001.