CNS-targeted base editing of the major late-onset Tay-Sachs mutation alleviates disease in mice
Maria L. Allende, Mari Kono, Y. Terry Lee, Samantha M. Olmsted, Vienna Huso, Jenna Y. Bakir, Florencia Pratto, Cuiling Li, Colleen Byrnes, Galina Tuymetova, Hongling Zhu, Cynthia J. Tifft, Richard L. Proia
Maria L. Allende, Mari Kono, Y. Terry Lee, Samantha M. Olmsted, Vienna Huso, Jenna Y. Bakir, Florencia Pratto, Cuiling Li, Colleen Byrnes, Galina Tuymetova, Hongling Zhu, Cynthia J. Tifft, Richard L. Proia
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Research Article Genetics Neuroscience

CNS-targeted base editing of the major late-onset Tay-Sachs mutation alleviates disease in mice

Abstract

Late-onset Tay-Sachs (LOTS) disease is a lysosomal storage disorder most commonly caused by a point mutation (c.805G>A) in the HEXA gene encoding the α subunit of the lysosomal enzyme β-hexosaminidase A. LOTS manifests as a range of gradually worsening neurological symptoms beginning in young adulthood. Here, we explored the efficacy of an adenine base editor (ABE) programmed with an sgRNA to correct the HEXA c.805G>A mutation. Base editing in fibroblasts from a patient with LOTS successfully converted the pathogenic HEXA c.805A to G and partially restored β-hexosaminidase activity, with minimal genome-wide off-target editing. We generated a LOTS mouse model in which the mice exhibited decreased β-hexosaminidase activity, accumulation of GM2 ganglioside in the brain, progressive neurological manifestations, and reduced lifespan. Treatment of LOTS mice with the neurotropic virus AAV-PHP.eB carrying the ABE and an sgRNA targeting the LOTS point mutation partially corrected the c.805G>A mutation in the CNS, significantly increased brain β-hexosaminidase activity, and substantially reduced GM2 ganglioside accumulation in the brain. Moreover, the therapy delayed symptom onset and significantly extended median lifespan. These findings highlight the potential of base editing as an effective treatment for LOTS and its broader applicability to other lysosomal storage disorders.

Authors

Maria L. Allende, Mari Kono, Y. Terry Lee, Samantha M. Olmsted, Vienna Huso, Jenna Y. Bakir, Florencia Pratto, Cuiling Li, Colleen Byrnes, Galina Tuymetova, Hongling Zhu, Cynthia J. Tifft, Richard L. Proia

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Figure 5

Base-editor treatment reduces brain expression of neuroinflammation markers in LOTS mice.

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Base-editor treatment reduces brain expression of neuroinflammation mark...
RNA-Seq was performed on brains from WT, control-treated LOTS, and ABE-treated LOTS male mice at 21 weeks of age (n = 4 per group). The AAV-treated mice each received 2.4 × 1012 vg. Brains were harvested, RNA was extracted, and transcriptome analysis was performed using the NovoMagic platform (Novogene). (A) Heatmap showing row z scores for the top 25 genes significantly differentially expressed between control-treated LOTS and WT mice. Each column represents an individual mouse. (B and C) Expression of astrocyte-related genes (B) and macrophage/microglia-related genes (C) shown as fragments per kilobase of transcript per million mapped reads (FPKM). Data are presented as mean ± SD; each dot represents 1 mouse. ***P < 0.001 (1-way ANOVA with Bonferroni’s correction). (D–G) Quantitative PCR validation of RNA-Seq results. Expression of CD68, GFAP, Gpnmb, and Lgals3bp mRNA in WT, control-treated, and ABE-treated mouse brains. n = 4 per group. Data are expressed as mean ± SD. **P < 0.01, ***P < 0.001, ****P < 0.0001 (1-way ANOVA with Bonferroni’s correction). (H–J) Western blot validation of protein expression. Representative blots (left) and quantification (right) for CD68 (H), GFAP (I), and Gpnmb (J) in brain extracts from WT, control-treated LOTS, and ABE-treated LOTS mice. β-Actin served as a loading control. n = 3 per group. Data are expressed as mean ± SD. **P < 0.01, ****P < 0.0001 (1-way ANOVA with Bonferroni’s correction).