Therapeutic efficacy of bone marrow transplant, intracranial AAV-mediated gene therapy, or both in the mouse model of MPS IIIB

Abstract

Sanfilippo syndrome type B (MPS IIIB) is a lysosomal storage disease resulting from a deficiency of N-acetyl-glucosaminidase (NAGLU) activity. In an attempt to correct the disease in the murine model of MPS IIIB, neonatal mice were treated with intracranial AAV2/5-NAGLU (AAV), syngeneic bone marrow transplant (BMT), or both (AAV/BMT). All treatments resulted in some improvement in clinical phenotype. Adeno-associated viral (AAV) treatment resulted in improvements in lifespan, motor function, hearing, time to activity onset, and daytime activity level, but no reduction of lysosomal storage. BMT resulted in improved hearing by 9 months, and improved circadian measures, but had no effect on lifespan, motor function, or central nervous system (CNS) lysosomal storage. AAV/BMT treatment resulted in improvements in hearing, time to activity onset, motor function, and reduced CNS lysosomal storage, but had no effect on lifespan. Combination therapy compared to either therapy alone resulted in synergistic effects on hearing and CNS lysosomal inclusions but antagonistic effects on motor function and lifespan. AAV alone is more efficacious than BMT or AAV/BMT treatment for lifespan. BMT was the least efficacious treatment by all measures. CNS-directed AAV treatment alone appears to be the preferred treatment, combining the most efficacy with the least toxicity of the approaches assessed.

Figure 1

Tissue NAGLU enzyme activity in treated and untreated mice. (a) Brain, (b) liver, (c) spleen, and (d) heart from 4 to 6 animals in each group were homogenized and N-acetyl-glucosaminidase activity was determined. Activity values ± SEM for each MPS IIIB group are graphed relative to the mean of the normal untreated group. Significant differences of treated from untreated MPS IIIB groups are indicated with *P < 0.05 and **P < 0.01. AAV, adeno-associated virus; BMT, bone marrow transplant; NAGLU, N-acetyl-glucosaminidase.

Figure 2

Tissue β-glucuronidase activity in treated and untreated mice. (a) Brain, (b) liver, and (c) spleen from 4 to 6 animals in each group were homogenized and β-glucuronidase activity was determined. Activity values ± SEM for each MPS IIIB group are graphed relative to the mean of the normal untreated group. Significant differences of treated from untreated MPS IIIB groups are indicated with *P < 0.05. AAV, adeno-associated virus; BMT, bone marrow transplant.

Figure 3

Circadian assessment of treated and untreated mice. Male mice from each group (n = 5 except for n = 3 for MPS IIIB-BMT) were individually housed in cages, and wheel running activity was recorded from ages 8 to 12 weeks. (a) Proportion of activity occurring while lights were on, and (b) mean time from lights off to activity onset are plotted ± SEM. AAV, adeno-associated virus; BMT, bone marrow transplant.

Figure 4

Hearing sensitivity in decibels of treated and untreated mice at 10 kHz. Auditory-evoked brainstem responses (ABR) recording was performed at 5, 10, 20, 28.3, and 40 kHz on 13–17 animals per group at 3, 6, and 9 months of age. The 10 kHz data are representative of the trends observed at each of the frequencies tested. Each group's mean decibel threshold to detect the audible stimuli is shown at 3, 6, and 9 months of age. Significant differences of treated from untreated MPS IIIB groups are indicated with *P < 0.05 and **P < 0.01. AAV, adeno-associated virus; BMT, bone marrow transplant.

Figure 5

Light photomicrographs of cochlea in representative mice from each treatment group. Within the lower base: (a) untreated MPS IIIB mouse (no Tx) shows loss of hair cell receptors (along with the entire organ of Corti, arrow), and loss of afferent neuronal processes (arrowheads). (b–d) AAV alone and AAV/BMT appear more protective of receptor cells and neurons than BMT alone. (e) Untreated normal (NL) mouse. Within the upper base: (f) untreated MPS IIIB mouse shows abundant storage in fibrocytes of the spiral ligament (white arrowheads). Storage is modestly reduced in mouse receiving (g) AAV alone and more apparently reduced by (h) AAV/BMT in combination. AAV, adeno-associated virus; BMT, bone marrow transplant; OC, organ of Corti.

Figure 6

Rocking rotarod performance as a function of age in each treatment group. Mean duration of the time spent on a rocking (reversing) rotarod as a function of increasing age for all treated and normal control groups is depicted. No performance deficits were evident in the untreated MPS IIIB mice (or in the other treated groups) compared to normal controls during the predysfunction period (168, 196, 224, and 252 days). However, a significant main effect of group (*P = 0.048) and a significant group by age interaction (**P = 0.008) suggest that age-dependent group performance differences occurred during the progressive impairment period (280, 308, 336, and 364 days). Pairwise comparisons showed that on average across the ages at testing, the MPS IIIB mice were significantly impaired relative to normal control mice (P = 0.004) and that MPS IIB-AAV mice showed improved performance compared to MPS IIB mice (P = 0.040). Subsequent one-way analysis of variances conducted at each age showed that these effects were mostly due to differences observed at 364 days (P = 0.005). At this age, the untreated MPS IIIB mice showed performance deficits compared to the normal controls (^†P = 0.0006), and MPS IIIB-AAV mice exhibited significantly improved performance levels relative to the untreated MPS IIIB mice (^††P = 0.010). AAV, adeno-associated virus; BMT, bone marrow transplant.

Figure 7

Survival curves by Kaplan–Meier (KM) analysis. All treated mice were analyzed for median survival by KM analysis and plots are shown. Normal-AAV/BMT did not differ from untreated normals and MPS IIIB-BMT did not differ from MPS IIIB, so these two groups are not shown for clarity. The MPS IIIB-AAV group had a significantly prolonged median survival compared to MPS IIIB (**P = 0.0054). AAV, adeno-associated virus; BMT, bone marrow transplant.