Repetitive, non-invasive imaging of neurodegeneration, and prevention of it with gene replacement, in mice with Sanfilippo syndrome.

Hampering assessment of treatment outcomes in gene therapy and other clinical trials in patients with childhood dementia is the lack of an objective, non-invasive measure of neurodegeneration. Optical coherence tomography (OCT) is a widely available, rapid, non-invasive, and quantitative method for examining the integrity of the neuroretina. Profound brain and retinal dysfunction occur in patients and animal models of childhood dementia, including Sanfilippo syndrome and we recently revealed a correlation between the age of onset and rate of progression of retinal and brain degeneration in sulfamidase-deficient Sanfilippo mice. The aim of the current study was to use OCT to visualise the discrete changes in retinal structure that occur during disease progression. A progressive decline in retinal thickness was readily observable in Sanfilippo mice using OCT, with differences seen in affected animals from 10-weeks of age. OCT applied to i.v. AAV9-sulfamidase-treated Sanfilippo mice enabled visualisation of improved retinal anatomy in living animals, an outcome confirmed via histology. Importantly, brain disease lesions were also ameliorated in treated Sanfilippo mice. The findings highlight the sensitivity, ease of repetitive use and quantitative capacity of OCT for detection of discrete changes in retinal structure and their prevention with a therapeutic. Combined with the knowledge that retinal and brain degeneration are correlated in Sanfilippo syndrome, OCT provides a window to the brain in this and potentially other childhood dementias.

Biomarkers for predicting disease course in Sanfilippo syndrome: An urgent unmet need in childhood-onset dementia.

Sanfilippo syndrome (MPS III) is an autosomal recessive inherited disorder causing dementia in children, following an essentially normal early developmental period. First symptoms typically include delayed language development, hyperactivity and/or insomnia from 2 years of age, followed by unremitting and overt loss of previously acquired skills. There are no approved treatments, and the median age of death is 18 years. Treatments under clinical trial demonstrate therapeutic benefit when applied pre-symptomatically in children diagnosed early through known familial inheritance risk. Newborn screening for Sanfilippo syndrome would enable pre-symptomatic diagnosis and optimal therapeutic benefit, however, many fold more patients with Sanfilippo syndrome are expected to be identified in the population than present with childhood dementia. Therefore, the capacity to stratify which Sanfilippo infants will need treatment in toddlerhood is necessary. While diagnostic methods have been developed, and continue to be refined, currently there are no tools or laboratory-based biomarkers available to provide pre-symptomatic prognosis. There is also a lack of progression and neurocognitive response-to-treatment biomarkers; disease stage and rate of progression are currently determined by age at symptom onset, loss of cerebral grey matter volume by magnetic resonance imaging and developmental quotient score for age. Robust blood-based biomarkers are an urgent unmet need. In this review, we discuss the development of biomarker assays for Sanfilippo based on the neuropathological pathways known to change leading into symptom onset and progression, and their performance as biomarkers in other neurodegenerative diseases. We propose that neural-derived exosomes extracted from blood may provide an ideal liquid biopsy to detect reductions in synaptic protein availability, and mitochondrial function. Furthermore, given the prominent role of neuroinflammation in symptom expression, glial fibrillary acidic protein detection in plasma/serum, alongside measurement of active brain atrophy by neurofilament light chain, warrant increased investigation for prognostic, progression and neurocognitive response-to-treatment biomarker potential in Sanfilippo syndrome and potentially other childhood dementias.

The ovine Type II Gaucher disease model recapitulates aspects of human brain disease.

Acute neuronopathic (type II) Gaucher disease (GD) is a devastating, untreatable neurological disorder resulting from mutations in the glucocerebrosidase gene (GBA1), with subsequent accumulation of glucosylceramide and glucosylsphingosine. Patients experience progressive decline in neurological function, with onset typically within the first three-to-six months of life and premature death before two years. Mice and drosophila with GD have been described, however little is known about the brain pathology observed in the naturally occurring ovine model of GD. We have characterised pathological changes in GD lamb brain and compared the histological findings to those in GD patient post-mortem tissue, to determine the validity of the sheep as a model of this disease. Five GD and five age-matched unaffected lamb brains were examined. We observed significant expansion of the endo/lysosomal system in GD lamb cingulate gyrus however TPP1 and cathepsin D levels were unchanged or reduced. H&E staining revealed neurons with shrunken, hypereosinophilic cytoplasm and hyperchromatic or pyknotic nuclei (red neurons) that were also shrunken and deeply Nissl stain positive. Amoeboid microglia were noted throughout GD brain. Spheroidal inclusions reactive for TOMM20, ubiquitin and most strikingly, p-Tau were observed in many brain regions in GD lamb brain, potentially indicating disturbed axonal trafficking. Our findings suggest that the ovine model of GD exhibits similar pathological changes to human, mouse, and drosophila type II GD brain, and represents a model suitable for evaluating therapeutic intervention, particularly in utero-targeted approaches.

AAVrh10 Vector Corrects Disease Pathology in MPS IIIA Mice and Achieves Widespread Distribution of SGSH in Large Animal Brains.

Patients with mucopolysaccharidosis type IIIA (MPS IIIA) lack the lysosomal enzyme sulfamidase (SGSH), which is responsible for the degradation of heparan sulfate (HS). Build-up of undegraded HS results in severe progressive neurodegeneration for which there is currently no treatment. The ability of the vector adeno-associated virus (AAV)rh.10-CAG-SGSH (LYS-SAF302) to correct disease pathology was evaluated in a mouse model for MPS IIIA. LYS-SAF302 was administered to 5-week-old MPS IIIA mice at three different doses (8.6E+08, 4.1E+10, and 9.0E+10 vector genomes [vg]/animal) injected into the caudate putamen/striatum and thalamus. LYS-SAF302 was able to dose-dependently correct or significantly reduce HS storage, secondary accumulation of GM2 and GM3 gangliosides, ubiquitin-reactive axonal spheroid lesions, lysosomal expansion, and neuroinflammation at 12 weeks and 25 weeks post-dosing. To study SGSH distribution in the brain of large animals, LYS-SAF302 was injected into the subcortical white matter of dogs (1.0E+12 or 2.0E+12 vg/animal) and cynomolgus monkeys (7.2E+11 vg/animal). Increases of SGSH enzyme activity of at least 20% above endogenous levels were detected in 78% (dogs 4 weeks after injection) and 97% (monkeys 6 weeks after injection) of the total brain volume. Taken together, these data validate intraparenchymal AAV administration as a promising method to achieve widespread enzyme distribution and correction of disease pathology in MPS IIIA.

Evaluation of Disease Lesions in the Developing Canine MPS IIIA Brain.

Mucopolysaccharidosis IIIA (MPS IIIA) is an inherited neurodegenerative disease of childhood that results in early death. Post-mortem studies have been carried out on human MPS IIIA brain, but little is known about early disease development. Here, we utilised the Huntaway dog model of MPS IIIA to evaluate disease lesion development from 2 to 24 weeks of age. A significant elevation in primarily stored heparan sulphate was observed in all brain regions assessed in MPS IIIA pups ≤9.5 weeks of age. There was a significant elevation in secondarily stored ganglioside (GM3 36:1) in ≤9.5-week-old MPS IIIA pup cerebellum, and other brain regions also exhibited accumulation of this lipid with time. The number of neural stem cells and neuronal precursor cells was essentially unchanged in MPS IIIA dog brain (c.f. unaffected) over the time course assessed, a finding corroborated by neuron cell counts. We observed early neuroinflammatory changes in young MPS IIIA pup brain, with significantly increased numbers of activated microglia recorded in all but one brain region in MPS IIIA pups ≤9.5 weeks of age (c.f. age-matched unaffected pups). In conclusion, infant-paediatric-stage MPS IIIA canine brain exhibits substantial and progressive primary and secondary substrate accumulation, coupled with early and robust microgliosis. Whilst early initiation of treatment is likely to be required to maintain optimal neurological function, the brain's neurodevelopmental potential appears largely unaffected by the disease process; further investigations confirming this are warranted.

A Preclinical Study Evaluating AAVrh10-Based Gene Therapy for Sanfilippo Syndrome.

Mucopolysaccharidosis type IIIA (MPS IIIA) is predominantly a disorder of the central nervous system, caused by a deficiency of sulfamidase (SGSH) with subsequent storage of heparan sulfate-derived oligosaccharides. No widely available therapy exists, and for this reason, a mouse model has been utilized to carry out a preclinical assessment of the benefit of intraparenchymal administration of a gene vector (AAVrh10-SGSH-IRES-SUMF1) into presymptomatic MPS IIIA mice. The outcome has been assessed with time, measuring primary and secondary storage material, neuroinflammation, and intracellular inclusions, all of which appear as the disease progresses. The vector resulted in predominantly ipsilateral distribution of SGSH, with substantially less detected in the contralateral hemisphere. Vector-derived SGSH enzyme improved heparan sulfate catabolism, reduced microglial activation, and, after a time delay, ameliorated GM3 ganglioside accumulation and halted ubiquitin-positive lesion formation in regions local to, or connected by projections to, the injection site. Improvements were not observed in regions of the brain distant from, or lacking connections with, the injection site. Intraparenchymal gene vector administration therefore has therapeutic potential provided that multiple brain regions are targeted with vector, in order to achieve widespread enzyme distribution and correction of disease pathology.

Delivery of therapeutic protein for prevention of neurodegenerative changes: comparison of different CSF-delivery methods.

Injection of lysosomal enzyme into cisternal or ventricular cerebrospinal fluid (CSF) has been carried out in 11 lysosomal storage disorder models, with each study demonstrating reductions in primary substrate and secondary neuropathological changes, and several reports of improved neurological function. Whilst acute studies in mucopolysaccharidosis (MPS) type II mice revealed that intrathecally-delivered enzyme (into thoraco-lumbar CSF) accesses the brain, the impact of longer-term treatment of affected subjects via this route is unknown. This approach is presently being utilized to treat children with MPS types I, II and III. Our aim was to determine the efficacy of repeated intrathecal injection of recombinant human sulfamidase (rhSGSH) on pathological changes in the MPS IIIA dog brain. The outcomes were compared with those in dogs treated via intra-cisternal or ventricular routes. Control dogs received buffer or no treatment. Significant reductions in primary/secondary substrate levels in brain were observed in dogs treated via all routes, although the extent of the reduction differed regionally. Treatment via all CSF access points resulted in large reductions in microgliosis in superficial cerebral cortex, but only ventricular injection enabled amelioration in deep cerebral cortex. Formation of glutamic acid decarboxylase-positive axonal spheroids in deep cerebellar nuclei was prevented by treatment delivered via any route. Anti-rhSGSH antibodies in the sera of some dogs did not reduce therapeutic efficacy. Our data indicates the capacity of intra-spinal CSF-injected rhSGSH to circulate within CSF-filled spaces, penetrate into brain and mediate a significant reduction in substrate accumulation and secondary pathology in the MPS IIIA dog brain.

Neonatal Bone Marrow Transplantation in MPS IIIA Mice.

Patients with some neurological lysosomal storage disorders (LSD) exhibit improved clinical signs following bone marrow transplantation (BMT). The failure of mucopolysaccharidosis (MPS) type IIIA patients and adult mice with the condition to respond to this treatment may relate to factors such as impaired migration of donor-derived cells into the brain, insufficient enzyme production and/or secretion by the donor-derived microglial cells, or the age at which treatment is initiated. To explore these possibilities, we treated neonatal MPS IIIA mice with whole unfractionated bone marrow and observed that nucleated blood cell reconstitution occurred to a similar degree in MPS IIIA mice receiving green fluorescent protein (GFP)-expressing normal (treatment group) or MPS IIIA-GFP marrow (control group) and normal mice receiving normal-GFP marrow (control group). Further, similar distribution patterns of GFP(+) normal or MPS IIIA donor-derived cells were observed throughout the MPS IIIA mouse brain. We demonstrate that N-sulfoglucosamine sulfohydrolase (SGSH), the enzyme deficient in MPS IIIA, is produced and secreted in a manner proportional to that of other lysosomal enzymes. However, despite this, overall brain SGSH activity was unchanged in MPS IIIA mice treated with normal marrow and the lysosomal storage burden in whole brain homogenates did not decrease, most likely due to donor-derived cells comprising <0.24% of total recipient brain cells in all groups. This suggests that the failure of MPS IIIA patients and mice to respond to BMT may occur as a result of insufficient donor-derived enzyme production and/or uptake by host brain cells.

Aldosterone glucuronidation by human liver and kidney microsomes and recombinant UDP-glucuronosyltransferases: inhibition by NSAIDs.

AIMS: To characterize: i) the kinetics of aldosterone (ALDO) 18beta-glucuronidation using human liver and human kidney microsomes and identify the human UGT enzyme(s) responsible for ALDO 18beta-glucuronidation and ii) the inhibition of ALDO 18beta-glucuronidation by non-selective NSAIDs. METHODS: Using HPLC and LC-MS methods, ALDO 18beta-glucuronidation was characterized using human liver (n= 6), human kidney microsomes (n= 5) and recombinant human UGT 1A1, 1A3, 1A4, 1A5, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B10, 2B15, 2B17 and 2B28 as the enzyme sources. Inhibition of ALDO 18beta-glucuronidation was investigated using alclofenac, cicloprofen, diclofenac, diflunisal, fenoprofen, R- and S-ibuprofen, indomethacin, ketoprofen, ketorolac, meclofenamic acid, mefenamic acid, S-naproxen, pirprofen and tiaprofenic acid. A rank order of inhibition (IC(50)) was established and the mechanism of inhibition investigated using diclofenac, S-ibuprofen, indomethacin, mefenamic acid and S-naproxen. RESULTS: ALDO 18beta-glucuronidation by hepatic and renal microsomes exhibited Michaelis-Menten kinetics. Mean (+/-SD) K(m), V(max) and CL(int) values for HLM and HKCM were 509 +/- 137 and 367 +/- 170 microm, 1075 +/- 429 and 1110 +/- 522 pmol min(-1) mg(-1), and 2.36 +/- 1.12 and 3.91 +/- 2.35 microl min(-1) mg(-1), respectively. Of the UGT proteins, only UGT1A10 and UGT2B7 converted ALDO to its 18beta-glucuronide. All NSAIDs investigated inhibited ALDO 18beta-G formation by HLM, HKCM and UGT2B7. The rank order of inhibition (IC(50)) of renal and hepatic ALDO 18beta-glucuronidation followed the general trend: fenamates > diclofenac > arylpropionates. CONCLUSION: A NSAID-ALDO interaction in vivo may result in elevated intra-renal concentrations of ALDO that may contribute to the adverse renal effects of NSAIDs and their effects on antihypertensive drug response.

Quantitative prediction of in vivo inhibitory interactions involving glucuronidated drugs from in vitro data: the effect of fluconazole on zidovudine glucuronidation.

AIMS: Using the fluconazole-zidovudine (AZT) interaction as a model, to determine whether inhibition of UDP-glucuronosyltransferase (UGT) catalysed drug metabolism in vivo could be predicted quantitatively from in vitro kinetic data generated in the presence and absence bovine serum albumin (BSA). METHODS: Kinetic constants for AZT glucuronidation were generated using human liver microsomes (HLM) and recombinant UGT2B7, the principal enzyme responsible for AZT glucuronidation, as the enzyme sources with and without fluconazole. K(i) values were used to estimate the decrease in AZT clearance in vivo. RESULTS: Addition of BSA (2%) to incubations decreased the K(m) values for AZT glucuronidation by 85-90% for the HLM (923 +/- 357 to 91 +/- 9 microm) and UGT2B7 (478-70 microm) catalysed reactions, with little effect on V(max). Fluconazole, which was shown to be a selective inhibitor of UGT2B7, competitively inhibited AZT glucuronidation by HLM and UGT2B7. Like the K(m), BSA caused an 87% reduction in the K(i) for fluconazole inhibition of AZT glucuronidation by HLM (1133 +/- 403 to 145 +/- 36 microm) and UGT2B7 (529 to 73 microm). K(i) values determined for fluconazole using HLM and UGT2B7 in the presence (but not absence) of BSA predicted an interaction in vivo. The predicted magnitude of the interaction ranged from 41% to 217% of the reported AUC increase in patients, depending on the value of the in vivo fluconazole concentration employed in calculations. CONCLUSIONS: K(i) values determined under certain experimental conditions may quantitatively predict inhibition of UGT catalysed drug glucuronidation in vivo.