Pronounced Therapeutic Benefit of a Single Bidirectional AAV Vector Administered Systemically in Sandhoff Mice

Abstract

The GM2 gangliosidoses, Tay-Sachs disease (TSD) and Sandhoff disease (SD), are fatal lysosomal storage disorders caused by mutations in the HEXA and HEXB genes, respectively. These mutations cause dysfunction of the lysosomal enzyme β-N-acetylhexosaminidase A (HexA) and accumulation of GM2 ganglioside (GM2) with ensuing neurodegeneration, and death by 5 years of age. Until recently, the most successful therapy was achieved by intracranial co-delivery of monocistronic adeno-associated viral (AAV) vectors encoding Hex alpha and beta-subunits in animal models of SD. The blood-brain barrier crossing properties of AAV9 enables systemic gene therapy; however, the requirement of co-delivery of two monocistronic AAV vectors to overexpress the heterodimeric HexA protein has prevented the use of this approach. To address this need, we developed multiple AAV constructs encoding simultaneously HEXA and HEXB using AAV9 and AAV-PHP.B and tested their therapeutic efficacy in 4- to 6-week-old SD mice after systemic administration. Survival and biochemical outcomes revealed superiority of the AAV vector design using a bidirectional CBA promoter with equivalent dose-dependent outcomes for both capsids. AAV-treated mice performed normally in tests of motor function, CNS GM2 ganglioside levels were significantly reduced, and survival increased by >4-fold with some animals surviving past 2 years of age.

Keywords: AAV9; GM2 gangliosidosis; Sandhoff disease; Tay-Sachs disease; gene therapy; intravenous delivery.

Graphical abstract

Figure 1

Systemic Treatment with Bicistronic AAV Vectors Extends the Survival of SD Mice (A) Design of AAV-Bic and AAV-P2I vectors. (B) Vector dosing and median survival for all groups. Kaplan-Meier survival curves for SD mice treated systemically with (C) AAV-PHP.B-Bic, (D) AAV-PHP.B-P2I, (E) AAV9-Bic, and (F) AAV9-P2I vector. Only one control group of untreated SD (UT) and normal (WT+HZ) mice was included in the experiment and their survival curves shown in all graphs (C–F) for comparison purposes. Log-rank (Mantel-Cox) test showed that survival of all treatment groups was significantly (p < 0.0001) increased compared to untreated SD controls.

Figure 2

Motor Performance, Strength, and Coordination of Treated SD Mice Remain Normal Until at Least 150 Days of Age The performance of AAV-treated SD mice and controls was assessed starting at 60 days of age until 150 days of age in the (A) accelerating rotarod test (4–40 rpm) and (B) inverted screen test. The performance of untreated (UT) SD mice declined rapidly after 90 days of age with the last testing point at 120 days of age. Data are represented as mean ± SD

Figure 3

Hexosaminidase Activity Is Restored in the CNS and Liver of Treated SD Mice Mice (n = 6) from the high-dose cohorts were used (4 × 10¹² vg for AAV9 vectors and 1 × 10¹² vg for AAV-PHP.B vectors). Hexosaminidase activity (fold normal) in (A) CNS and (B) liver using artificial substrates 4-MUG and 4-MUGS hydrolyzed by all isozymes (total hexosaminidase) and HexA, respectively. (C) Isozyme analysis in liver of AAV treated SD mice and controls show peaks of 4-MUG activity in fractions 3, 15, and 22 corresponding to HexB (ββ), HexA (αβ), and HexS (αα), respectively. Treatment groups were compared to untreated SD mice using one-way ANOVA with Dunnett’s multiple comparison test for each tissue: ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.

Figure 4

Reduction in GM2 Ganglioside Content in the CNS of Treated SD Mice GM2 ganglioside was quantified in cerebrum, cerebellum, brainstem, and spinal cord of SD mice in the high dose cohorts at 150 days of age or humane endpoint (<150 days of age) for untreated SD mice using LC-MS/MS mass spectrometry. Treatment groups were compared to untreated SD mice using two-way ANOVA with Dunnett’s multiple comparison test for each tissue: ∗∗p < 0.01; ∗∗∗∗p < 0.0001.

Figure 5

Reduction of Microglial Activation in Brain and Spinal Cord of SD Mice Treated with AAV9-Bic Vector (A) Microglial activation was assessed by immunofluorescence staining for CD68 (red) and DAPI to identify nuclei (blue) in brain and spinal cord sections from untreated SD mice (untreated KO), SD mice treated with AAV9-Bic vector (high dose), as well as normal controls (WT). (B) Quantification of CD68 staining intensity showed statistically significant reductions in AAV9-Bic treated mice compared (white bars) to untreated SD mice (black bars) in brainstem and spinal cord (p < 0.05), as well as a trend toward lower levels in thalamus (p = 0.07). No significant differences between groups, including normal controls (gray bars) were apparent in cortex and cerebellum). Abbreviations are as follows: CT, cortex; TH, thalamus; BS, brainstem; CB, cerebellum; SC, spinal cord. Scale bars, 150 μm. Two tailed t tests were used to determine statistical significance (∗p < 0.05).

Figure 6

Systemic Treatment with AAV9-Bic Vector: Maximum Effective Treatment Age and Minimum Effective Dose (A) SD mice were treated with 1 × 10¹² vg AAV9-Bic at 8 (n = 10), 10 (n = 9), and 12 weeks (n = 8) of age and survival followed until the humane endpoint. (B) Dose escalation study in 4-week-old SD mice treated at doses of 1 × 10¹¹ (n = 11), 3 × 10¹¹ (n = 8), and 1 × 10¹² vg (n = 4). Kaplan-Meier survival curves are shown for treatment groups compared to a common group of untreated (UT) SD mice (n = 6). Log-rank (Mantel-Cox) test was used to compare survival of treatment groups to that of untreated SD mice: ∗∗p < 0.01; ∗∗∗∗p < 0.0001.