Development of neuropathology in murine MPS IIIA and MPS VII and the effect of N-butyldeoxynojirimycin treatment on MPS IIIA mice.

The mucopolysaccharidoses (MPSs) are a family of heritable diseases caused by deficiencies in glycosaminoglycan (GAG) degrading lysosomal enzymes. GAGs accumulate in a range of tissues, resulting in diverse pathology that includes brain degeneration. The secondary accumulation of glycosphingolipids, specifically Gм₂ and Gм₃ gangliosides, occurs in the MPS brain. In MPS IIIA and MPS VII mouse models GAGs and gangliosides began to accumulate prior to the onset of behavioural changes. Gм₂ levels began to rise early, following the trend of GAG accumulation, and increased to 548% and 219% of normal levels in MPS IIIA and MPS VII respectively. Gм₃ levels began to rise later, reaching a peak of 484% and 313% of normal in MPS IIIA and MPS VII respectively. Given that brain Gм₂ and Gм₃ accumulation precedes behavioural deficits, it is possible that these gangliosides contribute to brain degeneration. Thus, gangliosides may be a target for the treatment of MPS brain disease. N-butyldeoxynojirimycin (NB-DNJ) is an iminosugar capable of crossing the blood brain barrier and reducing brain ganglioside synthesis, consequently deceasing overall brain Gм₂ and Gм₃ levels. NB-DNJ treatment of MPS IIIA mice decreased brain Gм₂ and Gм₃ levels in the short but not in the long term. Despite this, the innate fear response was restored and learning ability was equivalent to normal with both lengths of treatment. MPS IIIA mice treated with NB-DNJ also had a reduction in cytokine gene expression, astroglial activation and oxidation of inflammatory lipids. Whether MPS IIIA behavioural improvements were due to a delay in ganglioside accumulation with NB-DNJ treatment, or due to an anti-inflammatory function of NB-DNJ is not known. However, this thesis demonstrates that NB-DNJ can improve MPS brain dysfunction in the MPS IIIA mouse model and may be a potential therapy for CNS disease for children with MPS.Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 201

Gene silencing of EXTL2 and EXTL3 as a substrate deprivation therapy for heparan sulphate storing mucopolysaccharidoses

Neurological pathology is characteristic of the mucopolysaccharidoses (MPSs) that store heparan sulphate (HS) glycosaminoglycan (gag) and has been proven to be refractory to systemic therapies. Substrate deprivation therapy (SDT) using general inhibitors of gag synthesis improves neurological function in mouse models of MPS, but is not specific to an MPS type. We have investigated RNA interference (RNAi) as a method of targeting SDT to the HS synthesising enzymes, EXTL2 and EXTL3. Multiple shRNA molecules specific to EXTL2 or EXTL3 were designed and validated in a reporter gene assay, with four out of six shRNA constructs reducing expression by over 90%. The three EXTL2-specific shRNA constructs reduced endogenous target gene expression by 68, 32 and 65%, and decreased gag synthesis by 46, 50 and 27%. One EXTL3-specific shRNA construct reduced endogenous target gene expression by 14% and gag synthesis by 39%. Lysosomal gag levels in MPS IIIA and MPS I fibroblasts were also reduced by EXTL2 and EXTL3-specific shRNA. Incorporation of shRNAs into a lentiviral expression system reduced gene expression, and one EXTL2-specific shRNA reduced gag synthesis. These results indicate that deprivation therapy through shRNA-mediated RNAi has potential as a therapy for HS-storing MPSs