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 2

Generation of a LOTS mouse model for base editing.

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Generation of a LOTS mouse model for base editing. (A) Schematic of the ...
(A) Schematic of the engineered Hexa locus. Cas9-mediated targeting inserted human HEXA exon 7 and flanking intron sequences into the mouse genome to generate either the reference allele (HEXA c.805G, left) or LOTS allele (HEXAc.805A, right). Mouse sequences are in blue; human sequences in red. (B) Body-weight progression in female mice. Weekly weights were recorded for HEXA c.805G and HEXA c.805G/Neu3-KO (left) and HEXA c.805A and HEXA c.805A/Neu3-KO mice (right). Data are mean ± SD (n = 10–15). *P < 0.05, **P < 0.01 (Student’s t test). (C) Kaplan-Meier survival curves for HEXA c.805G and HEXA c.805G/Neu3-KO (left) and HEXA c.805A and HEXAc.805A/Neu3-KO mice (right). Combined sexes shown (n = 25–57). (D and E) GM2 ganglioside levels in brain. Gangliosides were extracted from female mice aged 24–26 weeks (n = 2–3/genotype) and analyzed by high-performance TLC. (D) Representative high-performance TLC plate; each lane contains 5% of gangliosides from 1 brain hemisphere. Arrows indicate ganglioside standards. (E) Quantification of GM2 band intensities (mean ± SD). Each dot represents data from 1 mouse. ****P < 0.0001 (Student’s t test). (F) GM2 degradation pathways. In humans (red box) and mice (blue box), β-hexosaminidase A converts GM2 to GM3. In mice only, NEU3 also degrades GM2 to GA2, bypassing β-hexosaminidase A. (G) Generation of control and LOTS mice. HEXA c.805G or c.805A mice were crossed with Neu3-KO mice to generate HEXA c.805G/Neu3-KO (control) and HEXA c.805A/Neu3-KO (LOTS) mice. Diagrams show expected GM2 degradation in each line. (H) Neurological evaluation. Mice were scored weekly on 6 criteria (left box) starting at 7 weeks. Mean ± SD shown (n = 16 LOTS, n = 27 controls; sexes combined). Partially created with BioRender.com.