Intracerebroventricular administration of a modified hexosaminidase ameliorates late-stage neurodegeneration in a GM2 mouse model

Abstract

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.

Fig 1. Engineered HexD3 homodimer.

A) HexD3 model based on crystal structures of human beta-hexosaminidase A and beta-hexosaminidase isoform B. B) Modification of the HexM α subunit to generate the chimeric α₃’ subunit contained in HexD3. In panel A, crystal structures of human β-hexosaminidase A (PDB ID: 2GJX) and β-hexosaminidase isoform B (PDB ID: 1NOU) were used to generate the model of HexD3 (https://www.wwpdb.org/). HexD3 retains the GM2AP binding interface of the native α-β heterodimer, the α-subunit enzyme active site, and a β-β homodimer interface of the HexM homodimer [4]. Panel B shows how the parent HexM modified α subunit was further engineered to retain a β domain M6P site. Domain I of the HexM α subunit was replaced with domain I of the native β subunit. The resulting chimeric α₃’ subunit contains phosphorylated glycans at position N84 compared to the glycosylated, but not phosphorylated, S51N/A53T site in subunit α. Two additional β glycosylation sites, N190/N142, were also included. GM2AP, ganglioside GM2 activator protein; HexA, β-hexosaminidase A; M6P, mannose-6-phosphate; PDB, Protein Data Bank.

Fig 2. CNS localization and peripheral lysosomal correction following Hex delivery by ICV.

A) LAMP2 quantification following doses of HexA or HexD3 in various brain ROIs. B) MS quantitation of concentrations of HexA and D3 in the whole brain hemisphere following ICV dosing. C) Overlay of NeuN and naphthol signal showing localization to neurons in the hippocampus as well as relative concentrations of protein in tissue. D) HexA or HexD3 activity as seen in liver by naphthol signal. E) Liver LAMP2 levels after 3 to 7 doses of Hex enzyme ICV. ns, not significant; * P <0.05 using an unpaired t-test. N = 3–5 mice/group. CNS, central nervous system; Het, heterozygote; HexA, β-hexosaminidase A; ICV, intracerebroventricular; KO, knockout; LAMP2, lysosomal associated membrane protein-2; MS, mass spectrometry; NeuN, neuronal nuclear protein; ROI, region of interest; WT, wild type; veh, vehicle.

Fig 3. ICV delivery of HexD3 enzyme reduces brain substrates and lysosome BMP similarly to HexA.

A) HexA and HexD3 dosing strategy. B) HexA and HexD3 enzyme activity levels found in brain following dosing. C) Brain levels of BMP (22:6) after Hex enzyme dosing. D) Brain levels of GM2, GA2, and GM3 gangliosides after Hex enzyme dosing. E) Brain levels of N-glycan metabolite A2G0’ after Hex enzyme dosing. ns, not significant; * P <0.05; ** P <0.01 using a one-way ANOVA. In panel A, brain was harvested for analysis 24 hours after the last dose. In panel B, Sidak’s ANOVA test was used to compare HexA and HexD3. ANOVA, analysis of variance; BMP, bis(monoacylglycero)phosphate; HexA, β-hexosaminidase A; ICV, intracerebroventricular; MUG, 4-methylumbelliferyl-2-acetamido-2-deoxy-β-d-glucopyranoside; MUGS, 4-methylumbelliferyl-2-acetamido-2-deoxy-β-d-glucopyranoside-6-sulfate; t₀, time zero; t_≤-2wks, 2 weeks prior to ICV infusion; QW, once weekly; veh, vehicle; WT, wild type.

Fig 4. HexD3 ICV can ameliorate late-stage disease progression.

A) Schematic showing ages when ICV treatment was started in relation to the targeted stages of disease progression. B) Survival curves demonstrating degree of lifespan benefit compared to untreated disease animals. C) Weight curves and D) nest building evaluating health in animals treated early and late with HexD3. E) Open-field locomotor activity after treatment with HexD3 and HexA beginning at various timepoints. F) Open-field locomotor activity when treatment was started after significant motor loss. ns, not significant; * P <0.05; ** P <0.01; *** P <0.001. N = 4–7 mice/group. For the UT group, n = 60; n ≥6 animals of mixed sex were enrolled per treatment group. In panel A, dotted and solid-colored lines represent HexA and HexD3 enzyme treatments, respectively; blue lines show degree of survival when treatment was started at 56 days of age; yellow lines show the same endpoint at 84 days of age; and red lines at 98 days of age. In panel A, differences between HexD3 and HexA were assessed with a log-rank Mantel-Cox test. In panel C, weight curves were assessed for males only, n ≥3. In panel D, nest building was assessed for mixed-sex groups, n ≥4. In panel E, open-field locomotor activity analysis was assessed in mixed-sex groups, n ≥6. In panel F, differences between vehicle and HexD3 were assessed with an unpaired t-test. For weight and distance, averages with standard deviation were plotted. For nest building, a violin plot was generated with median and quartile marks. All animals are Hexβ^-/- KO mice unless specified as Het, in which case Hexβ^+/- mice were used. Het, heterozygote; HexA, β-hexosaminidase A; ICV, intracerebroventricular; KO, knockout; UT, untreated; veh, vehicle; WT, wild type.