Enhanced survival in Sandhoff disease mice receiving a combination of substrate deprivation therapy and bone marrow transplantation

The G_M2 gangliosidoses are progressive, neurodegenerative lysosomal storage diseases.1 The hydrolysis of G_M2 is catalyzed by β-hexosaminidase. The HEXA and HEXB genes encode the β-hexosaminidase α and β subunits, respectively.1 Mutations in the α and β subunit genes result in Tay-Sachs and Sandhoff diseases, respectively. Potential therapeutic approaches include enzyme augmentation (enzyme replacement, bone marrow transplantation [BMT], or gene therapy) and substrate deprivation.2,3 In BMT microglial cells of donor origin are thought to repopulate the brain, becoming perivascular macrophages and microglia, and to supply enzyme to neurones by secretion–recapture.4-6 Substrate deprivation utilizes an inhibitor of GSL biosynthesis, such asN-butyldeoxynojirimycin (NB-DNJ).7By partially inhibiting GSL biosynthesis, the residual enzyme activity can hydrolyze the reduced influx of substrate into the lysosome, thus preventing storage. NB-DNJ inhibits the ceramide glucosyltransferase (glucosylceramide synthase, UDP-glucose–N-acylsphingosine D-glucosyltransferase, EC2.4.1.80), which is the first enzyme in the GSL biosynthetic pathway.3,8 In a mouse model of Tay-Sachs disease,NB-DNJ treatment reduced G_M2 accumulation in the brain.9 In the symptomatic mouse model of Sandhoff disease, NB-DNJ-treated mice10,11 survived 40% longer than untreated controls.10 BMT of Sandhoff mice extended life expectancy up to 8 months.12 Because the augmentation of enzyme through BMT and substrate deprivation may together show greater efficacy, we have treated Sandhoff mice with BMT and NB-DNJ and find that the 2 therapies act synergistically.

Sandhoff mice were bone marrow transplanted at 10 to 16 days of age.12 Drug treatment was carried out as previously described.10,13,NB-DNJ was a gift from Searle/Monsanto (St Louis, MO) and Oxford GlycoSciences (Abingdon, United Kingdom). Mice were tested on the bar crossing or inverted screen as previously described.10 The Open Field Test (box 45 × 25 × 12 cm with a 150 cm² floor grid) was conducted according to published methods.14 

We used published methods to assay β-hexosaminidase.10 BCA protein assay was used for protein determinations (Pierce, Chester, United Kingdom). GSL analysis was conducted as previously described.9 

Survival graphs were analyzed by the log-rank or the Mantel-Haenszel test.15 Log-likelihood test was used for β-hexosaminidase enzyme level correlations. We used the Studentt test to analyze β-hexosaminidase, GSL levels, and locomotion scores. Bar-crossing and inverted-screen test data were analyzed using a nonparametric regression model with a logistic link to the data set. P values were estimated using likelihood ratio. The statistical software used was S-PLUS version 3.4 (MathSoft, Seattle, WA).

Bone marrow–transplanted Sandhoff (SH) mice were treated withNB-DNJ (600 mg/kg per day) from 10 to 11 weeks of age. Four groups were studied: untreated (SH^UT), BMT monotherapy (SH^BMT), NB-DNJ monotherapy (SH^NB-DNJ), and combination therapy (BMT andNB-DNJ [SH^BMT/NB-DNJ]). Mean survival time (Figure 1A) of the SH^UTmice was 137 days. Survival was 166 days ± 4 (SH^NB-DNJ), 196 days ± 8 (SH^BMT), and 239 days ± 20 (SH^BMT/NB-DNJ). All treated mice had significantly (P < .001) increased lifespans compared with SH^UT mice (Figure 1A). SH^BMT/NB-DNJ mice survived significantly (P < .001) longer than SH^BMT or SH^NB-DNJ mice. The effect of NB-DNJ on BMT was 13% more than additive (P < .089, Figure 1A). When the SH^BMT mice were subdivided (50% of the mice into each category) on the basis of central nervous system β-hexosaminidase levels (2.26% ± 0.11% and 3.31% ± 0.94%, low- and high-enzyme groups, respectively; P = .108), the high-enzyme group exhibited 25% synergy (P < .001). The low-enzyme group exhibited an additive effect (P = .71) of NB-DNJ and BMT. Between the SH^BMT and SH^BMT/NB-DNJ groups, a similar level of β-hexosaminidase enzyme was measured in brain (P = .54) and spinal cord (P = .42) (Figure 1B) and in peripheral tissues (Figure 1C). In SH^BMT, there was no correlation between survival and β-hexosaminidase enzyme levels in brain, spinal cord, or peripheral tissues (P < .226). However, in the SH^BMT/NB-DNJ group, there was a significant correlation between survival and β-hexosaminidase levels in brain and spinal cord (P < .016, Pearson's correlation analysis) but not in liver, spleen, and kidney.

SH^BMT, SH^NB-DNJ, and SH^BMT/NB-DNJ groups differed from the SH^UTgroup in both the bar-crossing (P < .01) (Figure2A) and inverted-screen tests (P < .01) (Figure 2B). The SH^BMT group performed better in the bar-crossing test than the SH^NB-DNJgroup (P < .001), which performed better than the SH^UT group (P < .05). The SH^BMT/NB-DNJ group performed better than all other treatment groups (P < .023). The rate of decline in the SH^BMT/NB-DNJ group was significantly (P < .01) slower than in all other groups. In the inverted-screen test, performance started to decline at approximately 127 days for SH^UT but at approximately 140, 175, and 220 days for the SH^NB-DNJ, SH^BMT, and SH^BMT/NB-DNJ groups, respectively. The rates of decline were similar across groups. All groups displayed similar locomotor activity at 12 weeks, comparable to wild-type mice (Figure 2C). However, at 18 and 24 weeks of age, there were significant differences in activity between SH^UT and SH^NB-DNJ in the BMT (P < .01) and the non-BMT (P < .001) groups (Figure 2C).

G_M2 and G_A2 were measured at terminal stage of the disease (Table 1). In the brains of SH^NB-DNJ, SH^BMT, and SH^BMT/NB-DNJ mice, the G_A2 levels were comparable to those in SH^UT (P ≤ .478), even though they lived longer than the controls. All treated groups had the same G_A2 storage burden as the untreated mice, suggesting a slower rate of G_A2 accumulation. The brain G_M2levels were similar in the SH^NB-DNJ, SH^BMT, and SH^UT mice (P ≤ .614), but the SH^BMT/NB-DNJ mice had approximately 30% higher levels than the other groups (P ≤ .017). In spinal cord the G_M2 storage levels were comparable to those in brain. However, the spinal cord G_A2 levels were significantly lower in the SH^BMT (21%, P < .001) and in the SH^BMT/NB-DNJ (12%, P < .01) groups than in the SH^UT group. This may be due to higher levels of donor enzyme in the spinal cord (approximately 12% of normal; Figure1B). In the SH^NB-DNJ group, the liver G_M2 level was reduced by approximately 17% (P < .01) whereas the G_A2 level was comparable to that of the SH^UT(P = .26) at end stage. However, in the SH^BMTand SH^BMT/NB-DNJ groups, the liver GSL composition approximated that of the wild type because of high donor-enzyme levels (Figure 1C).

The onset of symptoms and the rates of disease progression were delayed significantly in SH^BMT/NB-DNJ relative to the monotherapy groups. By increasing the level of enzyme, the therapeutic response toNB-DNJ was enhanced. In brain, β-hexosaminidase activity from BMT was relatively small (approximately 2% to 5% of normal). The survival increase in the SH^BMT/NB-DNJ group was 13% more than the sum of the 2 monotherapy effects, indicating synergy. The survival of the combination group revealed 2 cohorts, those that survived the longest and those that died at an earlier time point. Survival correlated with the enzyme level in the brain. Synergy was approximately 25% for the high enzyme group compared to 13% for the group as a whole. The additional brain enzyme reconstitution needed to achieve approximately 25% synergy was only approximately 1.05% of the wild-type level. Mechanisms for the clinical benefit of BMT may extend beyond augmentation of enzyme levels. This may involve an inflammatory component with activated microglia contributing to neuronal dysfunction or death, as has been proposed for Alzheimer disease. The replacement of diseased microglial cells with wild-type cells by BMT, in combination with direct substrate reduction in neurones byNB-DNJ, may have led to synergy.

Glycosphingolipid analysis at terminal stage showed that for SH^BMT/NB-DNJ there was an increased storage burden of G_M2 and enhanced survival. This suggests that there is not a simple threshold level of brain G_M2 storage, which leads to disease. It supports a more complex mechanism of pathogenesis, as suggested previously.12 Levels of G_A2 are similar in all groups at their respective endpoints, suggesting that in the mouse there may be a threshold level of G_A2 that elicits disease. It is unclear why there is no elevated storage in the BMT group, despite their enhanced survival. After approximately 4 months it is possible that the rate of synthesis is lowered because of neuronal dysfunction, death, or both or that BMT only impacts the storage levels later in the disease course.

In conclusion, NB-DNJ therapy shows promise in treating GSL storage diseases, which involve neuropathology. These 2 therapeutic approaches act synergistically, provided that a high enough level of enzyme reconstitution can be achieved.

We thank Searle/Monsanto and Oxford GlycoSciences forNB-DNJ, Julia McAvoy and David Smith for excellent technical assistance, and Rob Deacon for his advice on behavioral tests.