Hematopoietic stem cell and gene therapy corrects primary neuropathology and behavior in mucopolysaccharidosis IIIA mice

Abstract

Mucopolysaccharidosis IIIA (MPS IIIA or Sanfilippo disease) is a neurodegenerative disorder caused by a deficiency in the lysosomal enzyme sulfamidase (SGSH), catabolizing heparan sulfate (HS). Affected children present with severe behavioral abnormalities, sleep disturbances, and progressive neurodegeneration, leading to death in their second decade. MPS I, a similar neurodegenerative disease accumulating HS, is treated successfully with hematopoietic stem cell transplantation (HSCT) but this treatment is ineffectual for MPS IIIA. We compared HSCT in MPS IIIA mice using wild-type donor cells transduced ex vivo with lentiviral vector-expressing SGSH (LV-WT-HSCT) versus wild-type donor cell transplant (WT-HSCT) or lentiviral-SGSH transduced MPS IIIA cells (LV-IIIA-HSCT). LV-WT-HSCT results in 10% of normal brain enzyme activity, near normalization of brain HS and GM2 gangliosides, significant improvements in neuroinflammation and behavioral correction. Both WT-HSCT and LV-IIIA-HSCT mediated improvements in GM2 gangliosides and neuroinflammation but were less effective at reducing HS or in ameliorating abnormal HS sulfation and had no significant effect on behavior. This suggests that HS may have a more significant role in neuropathology than neuroinflammation or GM2 gangliosides. These data provide compelling evidence for the efficacy of gene therapy in conjunction with WT-HSCT for neurological correction of MPS IIIA where conventional transplant is ineffectual.

Trial registration: ClinicalTrials.gov NCT01155778 NCT01299727.

Figures

Figure 1

Lentiviral N-sulfoglucosamine sulfohydrolase (SGSH) transduced microglia and hematopoietic stem cells (HSCs) have improved SGSH activity. (a) The lentiviral vector plasmid pHR'SIN-cPPT-SEW was converted to pHRsin.SFFV.hSGSH.att.wpre and pHRsin.SFFV.eGFP.att.wpre. These plasmids were used to produce lentiviral vector containing human SGSH or enhanced green fluorescent protein (eGFP) under the internal spleen focus-forming virus (SFFV) promoter. (b) The human microglial cell line CHME3 was transduced at an multiplicity of infection (MOI) of 10 with LV-SGSH or LV-eGFP and enzyme activity measured in cells and media. (c) Lineage depleted wild-type (WT) and mucopolysaccharidosis IIIA (MPS IIIA) bone marrow was transduced at an MOI of 25 with a lentiviral vector-expressing eGFP or SGSH, after 60 hours the enzyme activity was measured in cells and media. (d) Lineage depleted WT bone marrow was transduced at an MOI of 25 with a lentiviral vector-expressing eGFP or SGSH, after 14 days of culture in methylcellulose the number of colonies were counted. (d) The total number of colonies, (e) the percentage of the different colony types, and (f) the vector copy number was determined. (g) Lineage depleted MPS IIIA bone marrow was transduced at an MOI of 25 with a lentiviral vector-expressing eGFP or SGSH, after 14 days of culture in methylcellulose the number of colonies were counted. (g) The total number of colonies, (h) the percentage of the different colony types, and (i) the vector copy number was determined. Error bars represent the standard error of the mean (SEM) and significant difference is demonstrated with *P <0.05, **P <0.01 and ***P <0.001.

Figure 2

Lentiviral (LV)-N-sulfoglucosamine sulfohydrolase (SGSH) improves enzyme activity in the brain of mucopolysaccharidosis IIIA (MPS IIIA) mice. (a) Lineage depleted bone marrow was transduced or untransduced with a lentiviral vector-expressing human SGSH under the SFFV promoter and transplanted into busulfan conditioned recipients at 2 months (8 weeks) of age. Groups were; wild-type (WT) to WT transplant, WT untreated, MPS IIIA untreated, WT donor cells transduced with LV-SGSH into MPS IIIA recipients (LV-WT-HSCT), MPS IIIA donor cells transduced with LV-SGSH into MPS IIIA recipients (LV-IIIA-HSCT) and WT to MPS IIIA (WT-HSCT) (n = 16 per group). An open-field behavioral test was performed at 4 and 6 months (16 and 24 weeks) of age, chimerism and copy number were determined at 6 months of age and at 8 months of age (32 weeks of age, 24 weeks post-transplant), mice were sacrificed for biochemical and histological analysis and a cohort was kept for survival and sacrificed at their humane endpoint. (b) Donor chimerism was determined using flow cytometry at 16 weeks post-transplant. (c) Lentiviral vector copy number in white blood cells was determined by quantitative PCR (QPCR) at 16 weeks post-transplant. The average for the biochemistry/histology group (Figures 2–4 and Supplementary Figure S1), survival group (Figure 6) and overall/behavior group (Figure 5 and Supplementary Figure S2) has been shown. SGSH enzymatic activity was measured in (d) bone marrow (e) spleen, (f) liver, and (g) brain. Error bars represent the SEM. Significant difference to MPS IIIA is demonstrated with *P < 0.05, **P < 0.01, and ***P < 0.001. Where treatments result in significant improvement to MPS IIIA and there is no significant difference to WT, this is shown by a line and ns. Groups were; WT untreated (WT), WT donor cells transduced with LV-SGSH into MPS IIIA recipients (LV-WT-HSCT), MPS IIIA donor cells transduced with LV-SGSH into MPS IIIA recipients (LV-IIIA-HSCT), WT to MPS IIIA (WT-HSCT), and MPS IIIA untreated (MPS IIIA).

Figure 3

Lentiviral (LV)-N-sulfoglucosamine sulfohydrolase (SGSH) reduces primary storage in mucopolysaccharidosis IIIA (MPS IIIA) mice. At 8 months of age the level of sulfated glycosaminoglycans was determined using the Blyscan assay in the (a) liver and (b) brain. (c) The level of HS storage in the brain was determined by 2-aminoacridone (AMAC). (d) The different HS disaccharides were quantified. (e) The relative proportion of HS that was N-acetylated (NAc), N-sulfated (NS), 6-O-sulfated (6S), and 2-O-sulfated (2S) was also determined by AMAC. Error bars represent the SEM. Significant difference to MPS IIIA is demonstrated with *P < 0.05, **P < 0.01 and ***P < 0.001. Where treatments result in significant improvement to MPS IIIA and there is no significant difference to wild-type (WT), this is shown by a line and ns. Groups were; WT untreated (WT), WT donor cells transduced with LV-SGSH into MPS IIIA recipients (LV-WT-HSCT), MPS IIIA donor cells transduced with LV-SGSH into MPS IIIA recipients (LV-IIIA-HSCT), WT to MPS IIIA (WT-HSCT) and MPS IIIA untreated (MPS IIIA). The average copy number of the biochemistry/histology group was 0.47 integrations per white blood cell for LV-WT-HSCT and 0.11 integrations for LV-IIIA-HSCT groups.

Figure 4

Neuropathology is improved by all treatments. (a) Representative sections of brain cortex (layer IV/V) from –0.84 mm relative to bregma were stained with LAMP2 (green) to demonstrate lysosomal compartment size and DAPI (blue) to highlight nuclei. (Bar = 50 µm in low power and 100 µm in high power insert). (b) Representative images of cerebral cortex layer IV/V from around –0.46 mm relative to bregma showing isolectin B4-positive microglia (brown) and nuclei (blue) and (c) GM2 ganglioside (black). Two images of lentiviral (LV)-IIIA HSCT are shown, one with a high copy and one with a low copy to demonstrate the variable response in this group (Bar = 50 µm in low power and 100 µm in high power insert). (d) The number of microglia were counted, and (e) GM2 ganglioside storage was quantified using Image J from two fields of view (×20 objective) per brain section, four sections per mouse (n = 5 mice per group). Error bars represent the SEM. Significant difference to mucopolysaccharidosis IIIA (MPS IIIA) is demonstrated with *P < 0.05, **P < 0.01, and ***P < 0.001. Where treatments result in significant improvement to MPS IIIA and there is no significant difference to wild-type (WT) this is shown by a line and ns. Groups were; WT untreated (WT), WT donor cells transduced with LV-N-sulfoglucosamine sulfohydrolase (SGSH) into MPS IIIA recipients (LV-WT-HSCT), MPS IIIA donor cells transduced with LV-SGSH into MPS IIIA recipients (LV-IIIA-HSCT), WT to MPS IIIA (WT-HSCT) and MPS IIIA untreated (MPS IIIA). The representative high copy LV-IIIA-HSCT had a copy of 0.14 copies per white blood cell and the low copy contained 0.08 copies per white blood cell. The average copy number of the biochemistry/histology group was 0.47 integrations per white blood cell for LV-WT-HSCT and 0.11 integrations for LV-IIIA-HSCT groups.

Figure 5

Lentiviral (LV)-wild-type (WT)-HSCT corrects behavior. The open-field behavior test was performed for one hour at the same point of the circadian rhythm at 6 months (24 weeks) of age (n = 10 female mice per group). The measures of hyperactivity were: (a) path length, (b) duration spent moving over 100 mm/s, (c) frequency spent moving over 100 mm/s, (d) duration immobile, while (e) frequency of centre entries and (f) duration in centre measures thigmotaxis and may be a measure of sense of danger. Error bars represent the SEM. Significant difference to mucopolysaccharidosis IIIA (MPS IIIA) is demonstrated with *P < 0.05, **P < 0.01, and ***P < 0.001. Where treatments result in significant improvement to MPS IIIA and there is no significant difference to WT this is shown by a line and ns. Groups were; WT untreated (WT), WT donor cells transduced with LV-SGSH into MPS IIIA recipients (LV-WT-HSCT), MPS IIIA donor cells transduced with LV-SGSH into MPS IIIA recipients (LV-IIIA-HSCT), WT to MPS IIIA (WT-HSCT), and MPS IIIA untreated (MPS IIIA). The copy number of the behavioral cohort was 0.27 copies per white blood cell for LV-WT-HSCT and 0.25 for LV-IIIA-HSCT.

Figure 6

Lentiviral (LV)-N-sulfoglucosamine sulfohydrolase (SGSH) increases lifespan. A cohort of 6–10 mice from each group were kept to 92 weeks of age. Mice were sacrificed when they reached their humane end point, frequently caused due to urine retention. The estimated mean survival in weeks (w) for each group is shown at the bottom of the curve. The estimated mean survival was calculated by Mantel–Cox log rank pairwise comparisons, where data points were censored the largest survival time was used. Significant difference to mucopolysaccharidosis IIIA (MPS IIIA) is demonstrated with *P < 0.05, **P < 0.01, and ***P < 0.001 Groups were: wild-type (WT) untreated (WT), WT donor cells transduced with LV-SGSH into MPS IIIA recipients (LV-WT-HSCT), MPS IIIA donor cells transduced with LV-SGSH into MPS IIIA recipients (LV-IIIA-HSCT), WT to MPS IIIA (WT-HSCT) and MPS IIIA untreated (MPS IIIA). The copy number of the survival cohort was 0.16 copies per white blood cell for LV-WT-HSCT and 0.34 for LV-IIIA-HSCT.