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In vivo genome editing improves motor function and extends survival in a mouse model of ALS

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Science Advances  20 Dec 2017:
Vol. 3, no. 12, eaar3952
DOI: 10.1126/sciadv.aar3952
  • Fig. 1 In vivo genome editing reduces mutant SOD1 expression in G93A-SOD1 mice.

    (A) AAV vector schematic. ITR, inverted terminal repeat; NLS, nuclear localization signal sequence; 3×HA, three tandem repeats of the human influenza hemagglutinin (HA) epitope tag. (B) Schematic representation of the human SOD1 locus and the sgRNA target site. The arrowhead depicts approximate position of the G93A mutation. TSS, transcriptional start site; PAM, protospacer-adjacent motif. (C) Experimental timeline for in vivo studies. (D) Immunofluorescent staining of lumbar spinal cord sections and (E) Western blot of lumbar, thoracic, and cervical spinal cord lysate 4 weeks after G93A-SOD1 mice were injected with AAV9-SaCas9-hSOD1 (S; n = 3) and AAV9-SaCas9-mRosa26 (R; n = 3) via facial vein (quantitation of Western blot results in fig. S7). Arrowheads indicate ChAT+ and SaCas9+ cells with (upward) high or (downward) low hSOD1 expression. Images were captured using identical exposure conditions. Scale bar, 50 μm. (F) Indels from whole spinal cord tissue 4 weeks after G93A-SOD1 mice were injected with AAV9-SaCas9-hSOD1 (n = 3) via facial vein. Indels are colored dark green. Wild-type sequences are colored gray. The arrowhead indicates predicted SaCas9 cleavage site (D to F). All injections were performed at P0-P1.

  • Fig. 2 In vivo genome editing provides therapeutic benefit to G93A-SOD1 mice.

    (A and B) Disease onset, (C and D) survival, (E) rotarod performance, and (F) weight of G93A-SOD1 mice injected with AAV9-SaCas9-hSOD1 (n = 7), AAV9-SaCas9-mRosa26 (n = 7), or AAV9-EGFP (n = 7) via facial vein at P0-P1. (E and F) Mean rotarod times and weights were normalized to average 56-day values for each group. Wild type (n = 6) indicates litter-matching control mice. Values are means and error bars indicate (A to C) SD and (E and F) SEM. ***P < 0.0005; **P < 0.005; (A, C) one-way or (E and F) two-way analysis of variance (ANOVA) followed by Tukey’s post hoc analysis.

  • Fig. 3 G93A-SOD1 mice treated by genome editing have more ChAT+ cells at end stage compared to control mice.

    (A) Immunofluorescent staining of end-stage (top) lumbar or (bottom) thoracic spinal cord sections after G93A-SOD1 mice were injected with AAV9-SaCas9-hSOD1 or left untreated. (B) Mean number of ChAT+ neurons per end-stage (left) lumbar or (right) thoracic spinal cord hemisection after G93A-SOD1 mice were injected with AAV9-SaCas9-hSOD1 or left untreated (n = 4). Scale bars, 50 μm. Error bars indicate SD. *P < 0.05; **P < 0.001; two-tailed unpaired t test.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/3/12/eaar3952/DC1

    fig. S1. Designing sgRNA to target the human SOD1 gene.

    fig. S2. CRISPR-Cas9 reduced mutant SOD1 expression in NSC-34–G93A–SOD1 cells by genome editing.

    fig. S3. Quality control of AAV vectors.

    fig. S4. Mutant SOD1 expression in the spinal cord of untreated G93A-SOD1 mice.

    fig. S5. Systemic administration of AAV9-SaCas9-hSOD1 to neonatal G93A-SOD1 mice leads to SaCas9 expression in ChAT+ cells in the spinal cord.

    fig. S6. Systemic administration of AAV9-SaCas9-hSOD1 to neonatal G93A-SOD1 mice leads to SaCas9 expression in β3-tubulin+ fibers in the spinal cord.

    fig. S7. Systemic administration of AAV9-SaCas9-hSOD1 to neonatal G93A-SOD1 mice leads to limited SaCas9 expression in GFAP+ astrocytes in the spinal cord.

    fig. S8. CRISPR-Cas9–mediated genome editing reduced mutant SOD1 protein in G93A-SOD1 mice.

    fig. S9. Genome editing did not affect mouse SOD1 protein in G93A-SOD1 mice.

    fig. S10. Background modification at candidate OT sites in CRISPR-treated G93A-SOD1 mice.

    fig. S11. G93A-SOD1 mice treated with AAV9-SaCas9-hSOD1 lose weight at a slower rate after disease onset compared to control mice.

    fig. S12. Systemic administration of AAV9-SaCas9-hSOD1 to neonatal G93A-SOD1 mice did not delay the rate of disease progression.

    fig. S13. G93A-SOD1 mice injected with AAV9-SaCas9-SaCas9 had limited SaCas9 expression in GFAP+ astrocytes at end stage.

    fig. S14. Mutant SOD1 inclusion bodies were visible in end-stage spinal cord sections from CRISPR-treated G93A-SOD1 mice.

    table S1. Oligonucleotides used in this study.

    table S2. External primers for MiSeq analysis.

    table S3. Internal primers for MiSeq analysis.

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. Designing sgRNA to target the human SOD1 gene.
    • fig. S2. CRISPR-Cas9 reduced mutant SOD1 expression in NSC-34–G93A–SOD1 cells by genome editing.
    • fig. S3. Quality control of AAV vectors.
    • fig. S4. Mutant SOD1 expression in the spinal cord of untreated G93A-SOD1 mice.
    • fig. S5. Systemic administration of AAV9-SaCas9-hSOD1 to neonatal G93A-SOD1 mice leads to SaCas9 expression in ChAT+ cells in the spinal cord.
    • fig. S6. Systemic administration of AAV9-SaCas9-hSOD1 to neonatal G93A-SOD1 mice leads to SaCas9 expression in β3-tubulin+ fibers in the spinal cord.
    • fig. S7. Systemic administration of AAV9-SaCas9-hSOD1 to neonatal G93A-SOD1 mice leads to limited SaCas9 expression in GFAP+ astrocytes in the spinal cord.
    • fig. S8. CRISPR-Cas9–mediated genome editing reduced mutant SOD1 protein in G93A-SOD1 mice.
    • fig. S9. Genome editing did not affect mouse SOD1 protein in G93A-SOD1 mice.
    • fig. S10. Background modification at candidate OT sites in CRISPR-treated G93A-SOD1 mice.
    • fig. S11. G93A-SOD1 mice treated with AAV9-SaCas9-hSOD1 lose weight at a slower rate after disease onset compared to control mice.
    • fig. S12. Systemic administration of AAV9-SaCas9-hSOD1 to neonatal G93A-SOD1 mice did not delay the rate of disease progression.
    • fig. S13. G93A-SOD1 mice injected with AAV9-SaCas9-SaCas9 had limited SaCas9 expression in GFAP+ astrocytes at end stage.
    • fig. S14. Mutant SOD1 inclusion bodies were visible in end-stage spinal cord sections from CRISPR-treated G93A-SOD1 mice.
    • table S1. Oligonucleotides used in this study.
    • table S2. External primers for MiSeq analysis.
    • table S3. Internal primers for MiSeq analysis.

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