The GM2 gangliosidoses, Tay-Sachs disease and Sandhoff disease, are devastating neurodegenerative disorders caused by β-hexosaminidase A (HexA) deficiency. In the Sandhoff disease mouse model, rescue potential was severely reduced when HexA was introduced after disease onset. Here, we assess the effect of recombinant HexA and HexD3, a newly engineered mimetic of HexA optimized for the treatment of Tay-Sachs disease and Sandhoff disease. Enzyme replacement therapy was administered by repeat intracerebroventricular injections in Sandhoff disease model mice with dosing beginning before and after signs of neurodegeneration. As previously observed, HexA effectively increased the lifespan of Sandhoff disease mice by 3.5-fold only when treatment was started before onset of neurodegeneration. In contrast, HexD3 halted motor decline and ameliorated late-stage disease severity even when dosing began late, after neurodegeneration onset. Additionally, HexD3 had advantages over HexA in enzyme stability, distribution potential, and homodimer activity. Overall, our data indicate that advanced therapeutics may widen the treatment window for neurodegenerative disorders.

Krabbe disease (KD) and metachromatic leukodystrophy (MLD) are caused by accumulation of the glycolipids galactosylceramide (GalCer) and sulfatide and their toxic metabolites psychosine and lysosulfatide, respectively. We discovered a potent and selective small molecule inhibitor (S202) of ceramide galactosyltransferase (CGT), the key enzyme for GalCer biosynthesis, and characterized its use as substrate reduction therapy (SRT). Treating a KD mouse model with S202 dose-dependently reduced GalCer and psychosine in the central (CNS) and peripheral (PNS) nervous systems and significantly increased lifespan. Similarly, treating an MLD mouse model decreased sulfatides and lysosulfatide levels. Interestingly, lower doses of S202 partially inhibited CGT and selectively reduced synthesis of non-hydroxylated forms of GalCer and sulfatide, which appear to be the primary source of psychosine and lysosulfatide. Higher doses of S202 more completely inhibited CGT and reduced the levels of both non-hydroxylated and hydroxylated forms of GalCer and sulfatide. Despite the significant benefits observed in murine models of KD and MLD, chronic CGT inhibition negatively impacted both the CNS and PNS of wild-type mice. Therefore, further studies are necessary to elucidate the full therapeutic potential of CGT inhibition.

BMN 250 is being developed as enzyme replacement therapy for Sanfilippo type B, a primarily neurological rare disease, in which patients have deficient lysosomal alpha-N-acetylglucosaminidase (NAGLU) enzyme activity. BMN 250 is taken up in target cells by the cation-independent mannose 6-phosphate receptor (CI-MPR, insulin-like growth factor 2 receptor), which then facilitates transit to the lysosome. BMN 250 is dosed directly into the central nervous system via the intracerebroventricular (ICV) route, and the objective of this work was to compare systemic intravenous (IV) and ICV delivery of BMN 250 to confirm the value of ICV dosing. We first assess the ability of enzyme to cross a potentially compromised blood–brain barrier in the Naglu−/− mouse model and then assess the potential for CI-MPR to be employed for receptor-mediated transport across the blood–brain barrier. In wild-type and Naglu−/− mice, CI-MPR expression in brain vasculature is high during the neonatal period but virtually absent by adolescence. In contrast, CI-MPR remains expressed through adolescence in non-affected non-human primate and human brain vasculature. Combined results from IV administration of BMN 250 in Naglu−/− mice and IV and ICV administration in healthy juvenile non-human primates suggest a limitation to therapeutic benefit from IV administration because enzyme distribution is restricted to brain vascular endothelial cells: enzyme does not reach target neuronal cells following IV administration, and pharmacological response following IV administration is likely restricted to clearance of substrate in endothelial cells. In contrast, ICV administration enables central nervous system enzyme replacement with biodistribution to target cells.

Sanfilippo syndrome type B, or mucopolysaccharidosis IIIB (MPS IIIB), is a rare autosomal recessive lysosomal storage disease caused by a deficiency of alpha-N-acetylglucosaminidase (NAGLU). Deficiency in NAGLU disrupts the lysosomal turnover of heparan sulfate (HS), which results in the abnormal accumulation of partially degraded HS in cells and tissues. BMN 250 (NAGLU-insulin-like growth factor 2 [IGF2]) is a recombinant fusion protein developed as an investigational enzyme replacement therapy for MPS IIIB. The IGF2 peptide on BMN 250 promotes enhanced targeting of the enzyme to lysosomes through its interaction with the mannose 6-phosphate receptor. The focus of these studies was to further characterize the ability of NAGLU-IGF2 to clear accumulated HS compared to unmodified NAGLU in primary cellular models of MPS IIIB. Here, we establish distinct primary cell models of MPS IIIB with HS accumulation. These cellular models revealed distinct NAGLU uptake characteristics that depend on the duration of exposure. We found that with sustained exposure, NAGLU uptake and HS clearance occurred independent of known lysosomal targeting signals. In contrast, under conditions of limited exposure duration, NAGLU-IGF2 was taken up more rapidly than the unmodified NAGLU into MPS IIIB primary fibroblasts, astrocytes, and cortical neurons, where it efficiently degraded accumulated HS. These studies illustrate the importance of using physiologically relevant conditions in the evaluation of enzyme replacement therapies in cellular models.

Infantile globoid cell leukodystrophy (GLD, Krabbe disease) is a fatal demyelinating disorder caused by a deficiency in the lysosomal enzyme galactosylceramidase (GALC). GALC deficiency leads to the accumulation of the cytotoxic glycolipid, galactosylsphingosine (psychosine). Complementary evidence suggested that psychosine is synthesized via an anabolic pathway. Here, we show instead that psychosine is generated catabolically through the deacylation of galactosylceramide by acid ceramidase (ACDase). This reaction uncouples GALC deficiency from psychosine accumulation, allowing us to test the long-standing "psychosine hypothesis." We demonstrate that genetic loss of ACDase activity (Farber disease) in the GALC-deficient mouse model of human GLD (twitcher) eliminates psychosine accumulation and cures GLD. These data suggest that ACDase could be a target for substrate reduction therapy (SRT) in Krabbe patients. We show that pharmacological inhibition of ACDase activity with carmofur significantly decreases psychosine accumulation in cells from a Krabbe patient and prolongs the life span of the twitcher (Twi) mouse. Previous SRT experiments in the Twi mouse utilized L-cycloserine, which inhibits an enzyme several steps upstream of psychosine synthesis, thus altering the balance of other important lipids. Drugs that directly inhibit ACDase may have a more acceptable safety profile due to their mechanistic proximity to psychosine biogenesis. In total, these data clarify our understanding of psychosine synthesis, confirm the long-held psychosine hypothesis, and provide the impetus to discover safe and effective inhibitors of ACDase to treat Krabbe disease.

Sanfilippo syndrome type B (Sanfilippo B; Mucopolysaccharidosis type IIIB) occurs due to genetic deficiency of lysosomal alpha-N-acetylglucosaminidase (NAGLU) and subsequent lysosomal accumulation of heparan sulfate (HS), which coincides with devastating neurodegenerative disease. Because NAGLU expressed in Chinese hamster ovary cells is not mannose-6-phosphorylated, we developed an insulin-like growth factor 2 (IGF2)-tagged NAGLU molecule (BMN 250; tralesinidase alfa) that binds avidly to the IGF2 / cation-independent mannose 6-phosphate receptor (CI-MPR) for glycosylation independent lysosomal targeting. BMN 250 is currently being developed as an investigational enzyme replacement therapy for Sanfilippo B. Here we distinguish two cellular uptake mechanisms by which BMN 250 is targeted to lysosomes. In normal rodent-derived neurons and astrocytes, the majority of BMN250 uptake over 24 hours reaches saturation, which can be competitively inhibited with IGF2, suggestive of CI-MPR-mediated uptake. Kuptake, defined as the concentration of enzyme at half-maximal uptake, is 5 nM and 3 nM in neurons and astrocytes, with a maximal uptake capacity (Vmax) corresponding to 764 nmol/hr/mg and 5380 nmol/hr/mg, respectively. Similar to neurons and astrocytes, BMN 250 uptake in Sanfilippo B patient fibroblasts is predominantly CI-MPR-mediated, resulting in augmentation of NAGLU activity with doses of enzyme that fall well below the Kuptake (5 nM), which are sufficient to prevent HS accumulation. In contrast, uptake of the untagged recombinant human NAGLU (rhNAGLU) enzyme in neurons, astrocytes and fibroblasts is negligible at the same doses tested. In microglia, receptor-independent uptake, defined as enzyme uptake resistant to competition with excess IGF2, results in appreciable lysosomal delivery of BMN 250 and rhNAGLU (Vmax = 12,336 nmol/hr/mg and 5469 nmol/hr/mg, respectively). These results suggest that while receptor-independent mechanisms exist for lysosomal targeting of rhNAGLU in microglia, BMN 250, by its IGF2 tag moiety, confers increased CI-MPR-mediated lysosomal targeting to neurons and astrocytes, two additional critical cell types of Sanfilippo B disease pathogenesis.

Sanfilippo syndrome type B (mucopolysaccharidosis IIIB), caused by inherited deficiency of alpha-N-acetylglucosaminidase (NAGLU), required for lysosomal degradation of heparan sulfate (HS), is a pediatric neurodegenerative disorder with no approved treatment. Intracerebroventricular (ICV) delivery of a modified recombinant NAGLU, consisting of human NAGLU fused with insulin-like growth factor 2 (IGF2) for enhanced lysosomal targeting, was previously shown to result in marked enzyme uptake and clearance of HS storage in the Naglu(-/-) mouse brain. To further evaluate regional, cell type-specific, and dose-dependent biodistribution of NAGLUIGF2 (BMN 250) and its effects on biochemical and histological pathology, Naglu(-/-) mice were treated with 1-100 mu g ICV doses (four times over 2 weeks). 1 day after the last dose, BMN 250 (100 mu g doses) resulted in above-normal NAGLU activity levels, broad biodistribution, and uptake in all cell types, with NAGLU predominantly localized to neurons in the Naglu(-/-) mouse brain. This led to complete clearance of disease-specific HS and reduction of secondary lysosomal defects and neuropathology across various brain regions lasting for at least 28 days after the last dose. The substantial brain uptake of NAGLU attainable by this highest ICV dosage was required for nearly complete attenuation of disease-driven storage accumulations and neuropathology throughout the Naglu(-/-) mouse brain.

and only these have osteonecrosis; 5 received ERT at some time; age (range) diagnosed PD=56.4 (43-72) years; first PD signwas tremor in 9 (64.3%); cognitive dysfunction in all 14 GD+ PD. In GD+ PD, there was nearly twice larger Odds Ratio (OR) for the AG+ GG genotype (9) as for AA (5) while in control GD patients the AG+ GG versus AA value was equality (7). It has been shown that the OR of risk for PD increases with the number of minor alleles (with possible interactions with other SNPs at the same gene location and with other PD-associated SNCA SNPs at other locations). In the current study, this comparison was not greater among GD+ PD than amongmatched patients with GD only. This study showed a similar distribution regarding allele frequency so that the number of patients with at least oneminor allele (AG and GG) relative to those with only the major alleles (AA) was not greater among the GD+ PD compared to matched GD only patients. Similarly, in the aggregate, there was no important difference between the cohorts for frequencyof either allele singly (AversusG). The critical limitation of this study was the small number of GD+ PD patients involved, albeit including virtually the entire cohort of GD+ PD patients among the N500 adult type I GD patients in our clinic. Nonetheless, as a foray into the potential genetic susceptibility of patients with GD for a synucleinopathy or Lewy-body-like disease, this paradigm of casecontrols did not implicate a higher risk than in the general population.

Poly(ADP-ribose) polymerases 1 and 2 (PARP1 and PARP2), which are involved in DNA damage response, are targets of anticancer therapeutics. BMN 673 is a novel PARP1/2 inhibitor with substantially increased PARP-mediated tumor cytotoxicity and is now in later-stage clinical development for BRCA-deficient breast cancers. In co-crystal structures, BMN 673 is anchored to the nicotinamide-binding pocket via an extensive network of hydrogen-bonding and π-stacking interactions, including those mediated by active-site water molecules. The novel di-branched scaffold of BMN 673 extends the binding interactions towards the outer edges of the pocket, which exhibit the least sequence homology among PARP enzymes. The crystallographic structural analyses reported here therefore not only provide critical insights into the molecular basis for the exceptionally high potency of the clinical development candidate BMN 673, but also new opportunities for increasing inhibitor selectivity.

Significance Mucopolysaccharidosis type IIIB (MPS IIIB) is a devastating and currently untreatable disease affecting mainly the brain. The cause is lack of the lysosomal enzyme, α– N -acetylglucosaminidase (NAGLU), and storage of heparan sulfate. Using a mouse model of MPS IIIB, we administered a modified NAGLU by injection into the left ventricle of the brain, bypassing the blood–brain barrier. The modification consisted of a fragment of IGFII, which allows receptor-mediated uptake and delivery to lysosomes. The modified enzyme was taken up avidly by cells in both brain and liver, where it reduced pathological accumulation of heparan sulfate and other metabolites to normal or near-normal levels. The results suggest the possibility of treatment for MPS IIIB.