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. 2018 Oct 26:12:47-57.
doi: 10.1016/j.omtm.2018.10.011. eCollection 2019 Mar 15.

Efficacy of a Bicistronic Vector for Correction of Sandhoff Disease in a Mouse Model

Evan Woodley  1 , Karlaina J L Osmon  2 , Patrick Thompson  1 , Christopher Richmond  1 , Zhilin Chen  3 , Steven J Gray  4   5 , Jagdeep S Walia  1   2   3
Affiliations

Efficacy of a Bicistronic Vector for Correction of Sandhoff Disease in a Mouse Model

Evan Woodley et al. Mol Ther Methods Clin Dev. .

Abstract

GM2 gangliosidoses are a family of severe neurodegenerative disorders resulting from a deficiency in the β-hexosaminidase A enzyme. These disorders include Tay-Sachs disease and Sandhoff disease, caused by mutations in the HEXA gene and HEXB gene, respectively. The HEXA and HEXB genes are required to produce the α and β subunits of the β-hexosaminidase A enzyme, respectively. Using a Sandhoff disease mouse model, we tested for the first time the potential of a comparatively lower dose (2.04 × 1013 vg/kg) of systemically delivered single-stranded adeno-associated virus 9 expressing both human HEXB and human HEXA cDNA under the control of a single promoter with a P2A-linked bicistronic vector design to correct the neurological phenotype. A bicistronic design allows maximal overexpression and secretion of the Hex A enzyme. Neonatal mice were injected with either this ssAAV9-HexB-P2A-HexA vector or a vehicle solution via the superficial temporal vein. An increase in survival of 56% compared with vehicle-injected controls and biochemical analysis of the brain tissue and serum revealed an increase in enzyme activity and a decrease in brain GM2 ganglioside buildup. This is a proof-of-concept study showing the "correction efficacy" of a bicistronic AAV9 vector delivered intravenously for GM2 gangliosidoses. Further studies with higher doses are warranted.

Keywords: AAV9; Sandhoff disease; Tay-Sachs disease; gene therapy; hexosaminidase.

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Figures

Figure 1
Figure 1
ssAAV9-HexB-P2A-HexA Construct Design The human HEXA and HEXB genes were synthesized with a self-cleaving linker, P2A, between them and with a CAG promoter upstream to drive expression. The entire construct was designed between the appropriate AAV inverted terminal repeat (ITR) elements, and an SV40 element was included to promote protein expression in mammalian cells.
Figure 2
Figure 2
Hexosaminidase Activity and Protein Expression in HEKHexABKO Cells Transfected with the HexB-HexA Construct (A) MUG substrate was used to assess the hexosaminidase activity of cell lysates following DEAE column separation. ABKO cells showed no hexosaminidase activity above baseline, whereas WT HEK293 cells showed activity of all 3 isoforms of hexosaminidase. Following transfection with HexB-HexA, ABKO cells showed levels of hex activity equal to or greater than the WT HEK293 cells in all eluted fractions. (B) Protein expression analysis was conducted through western blot analysis, probing with anti-HEXA and anti-HEXB antibodies. Un-transfected HEKHexABKO cells showed no mature HexA or HexB expression, whereas ABKO cells transfected with the HexB-HexA construct (h.B2A plasmid) showed mature HexA and HexB expression levels.
Figure 3
Figure 3
Kaplan-Meier Survival Curve of the AAV-HexB-HexA and Drug-Treated Groups Shown is survival of mice over 32 weeks. Each point represents the percentage of mice surviving at that time. AAV-HexB-HexA treated-mice survived to a mean of 170 days, ranging from 146 to 186 days (n = 6). This extension in survival is significant compared with the untreated vehicle-injected SD mice with a mean of 110 days (p = 0.0002). All Het mice reached the study endpoint of 224 days. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Figure 4
Figure 4
Open Field Test Behavioral Analysis of AAV HexB-HexA and Drug-Treated Mice Shown is time moving during the 5-min open field test. Each point represents the mean time moving measured on a bi-weekly basis beginning at week 8. HexB-HexA-treated mice (n = 6) had a significantly higher mean time moving at 14 and 16 weeks of age compared with SD controls (n = 6). Het mice (n = 6) performed no better than HexB-HexA-treated SD mice until 24 weeks. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.
Figure 5
Figure 5
Brain Sectioning After extraction, the brain was roughly divided into the rostral brain (RB), middle brain (MB), and caudal brain (CB) sections. A 2-mm portion of the midbrain was extracted for histological processing. The remaining RB, MB, and CB slices were then halved, and each half was stored for further qPCR or biochemical analysis.
Figure 6
Figure 6
Biochemical Analysis of the HexB-HexA Treatment (A) Ganglioside storage analysis. Shown is densitometry-based quantification of GM2 ganglioside storage in the midbrain. HexB-HexA-treated mice (n = 6) showed a significantly lower proportion of GM2 ganglioside than SD controls (n = 6). Het controls showed no accumulation of the GM2 ganglioside. (B) Analysis of ganglioside content. Gangliosides isolated from frozen brains as well as standards were separated by high performance thin-layer chromatography (HPTLC) and detected with orcinol. Samples from brains of each cohort were examined. Representative samples are shown here; changes in the GM2 ganglioside band can be observed. (C) Analysis of Hex A activity by MUGS assay. No significant alterations in enzyme activity were observed in the treatment group compared with SD controls. (D) Serum collected at 8 weeks was assayed for Hex A activity using MUG substrate. HexB-HexA-treated SD mice (n = 6) showed significantly increased Hex A activity compared with SD controls (n = 6), as indicated by an increased breakdown of the substrate, resulting in an increased fluorescent signal (p = 0.0045). (E) Serum collected at 6 weeks was processed to separate hexosaminidase isoenzymes through DEAE columns and was collected in fractions. Enzyme activity in 3 separate HexB-HexA-treated mice showed increased hexosaminidase activity compared with pooled SD controls in a pattern that mirrored pooled Het control serum. These results include increased activity in fractions containing the Hex A enzyme (1–3) and the Hex B enzyme (9–12). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Figure 7
Figure 7
Histological Ganglioside Storage in Neurons of the Murine Midbrain at 16 Weeks Shown are sections of the murine cortex, hippocampus, hypothalamus, and thalamus from SD controls, HexB-HexA-treated SD mice, and Het controls. The black arrows point to GM2 ganglioside-filled neurons, which were found predominately in untreated SD mice and with reduced number and severity in the HexB-HexA-treated SD mice. A reduction in storage of GM2 ganglioside was observed in HexB-HexA-treated mice; however, it is not a full clearance of the GM2 gangliosides. No GM2 ganglioside storage is observed in the Het control sections. Scale bars represent 50 μm.
Figure 8
Figure 8
Vector Biodistribution of the hHEXB-P2A-HEXA Vector Viruses were distributed in most of the organs in long-term cohort and short-term cohort mice (n = 6 each). hHexBP2AHexA was not detectable above 0.000 copies per mouse genome in mice dosed with vehicle and is not shown in the graph. Data are presented as the copies of vector DNA per diploid mouse genome found in each assessed organ.
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