Research ArticleHEALTH AND MEDICINE

A mutation-independent CRISPR-Cas9–mediated gene targeting approach to treat a murine model of ornithine transcarbamylase deficiency

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Science Advances  12 Feb 2020:
Vol. 6, no. 7, eaax5701
DOI: 10.1126/sciadv.aax5701
  • Fig. 1 In vivo gene targeting of the OTC locus in the OTC spfash mouse liver by AAV.SaCas9.

    Schematic diagrams of the mouse OTC locus showing the SaCas9 target site located in intron 4, the AAV donor vector that contains U6-sgRNA1 and TBG-hOTCco-pA cassettes flanked by homology arms, and the modified mouse OTC locus after homologous recombination. ITR, inverted terminal repeat.

  • Fig. 2 Efficient and sustained expression of OTC in the liver of spfash mice treated as newborns with AAV8.SaCas9-mediated gene targeting.

    AAV8.SaCas9 (5 × 1010 GC per pup) and AAV8.sgRNA1.hOTCco donor (5 × 1011 GC per pup) were administrated to p2 spfash pups via the temporal vein. spfash mice were euthanized at 3 (targeted, 3 weeks; n = 6) or 8 weeks (targeted, 8 weeks; n = 8) after treatment. Untargeted spfash mice received AAV8.SaCas9 (5 × 1010 GC per pup) and AAV8.control.hOTCco donor (5 × 1011 GC per pup) at p2, and livers were harvested at 3 (untargeted, 3 weeks; n = 5) or 8 weeks (untargeted, 8 weeks; n = 8] after treatment. Untreated WT (n = 8) and spfash mice (n = 8) were included as controls. (A) Immunofluorescence staining with antibodies against OTC on liver sections from spfash mice treated with the dual AAV vectors for CRISPR-SaCas9–mediated gene targeting. Stained areas in the targeted groups typically represent clusters of OTC-expressing hepatocytes. Scale bar, 200 μm. (B) Histochemical staining of OTC enzyme activity on liver sections from spfash mice treated with the dual AAV vectors for CRISPR-SaCas9–mediated gene targeting. Scale bar, 200 μm. (C) Double immunofluorescence staining with antibodies against OTC (red) and glutamine synthetase (green), which is a marker of central veins. Scale bar, 200 μm. (D) Quantification of OTC-expressing cells based on the percentage of area on liver sections expressing OTC by immunostaining as presented in (A). (E) Quantification of hepatocytes expressing functional OTC based on the percentage of area on liver sections expressing OTC as presented in (B). (F) OTC enzyme activity in the liver homogenates of spfash mice at 3 and 8 weeks following dual vector treatment. (G) Quantification of hOTCco donor vector genome in the liver by quantitative PCR. ns, not statistically significant. *P < 0.05, **P < 0.01, ***P < 0.001, Mann-Whitney test.

  • Fig. 3 Functional improvement following high-protein diet challenge in spfash mice treated with the dual vectors for gene targeting.

    Seven weeks following neonatal treatment with the dual AAV vectors, mice were given high-protein diet for 7 days. (A) Plasma ammonia levels were measured 7 days after the high-protein diet. Plasma ammonia levels in WT mice (n = 10) and AAV8.SaCas9 + AAV8.sgRNA1.hOTCco donor-treated spfash mice (n = 12) were significantly lower than in untreated spfash mice (n = 15) after a 7-day high-protein diet. Red squares indicate samples obtained from moribund untreated spfash mice before the scheduled day 7 bleed; red triangles indicate sample obtained from a moribund spfash mice treated with untargeted vector (AAV8.control.hOTCco donor with no sgRNA1, n = 13) before the scheduled day 7 bleed. ns, not statistically different; *P < 0.05, ***P < 0.001, one-way analysis of variance (ANOVA), Dunnett’s test. (B) Survival curves in control or dual AAV vector–treated spfash mice after a 7-day course of high-protein diet. Untreated spfash mice (n = 18) or spfash mice treated with untargeted vectors (AAV8.control.hOTCco donor, n = 17) started to die 4 days after the high-protein diet. All WT (n = 10) and AAV8.SaCas9 + AAV8.sgRNA1.hOTCco donor-treated spfash mice (n = 15) survived. P < 0.05, log-rank test.

  • Fig. 4 Indel and HDR-mediated gene targeting efficiency analyses.

    Liver DNA was isolated from spfash mice 8 weeks after neonatal treatment with the dual gene-targeting vectors (n = 8) or untargeted vectors (n = 8). DNA from an untreated spfash mouse served as control. (A) Indel analysis on the targeted mOTC locus by deep sequencing. (B) HDR-mediated gene targeting efficiency analysis by LMU-PCR following digestion with Hae III or TaqI (see fig. S2). Means ± SEM are shown.

  • Fig. 5 Time course of OTC expression in liver by neonatal gene therapy, CRISPR-Cas9–mediated gene correction, or gene targeting.

    p2 spfash mice received temporal vein injection of AAV8.SaCas9 (5 × 1010 GC per pup) and 5 × 1011 GC per pup of AAV8.sRNA1.donor vector for CRISPR-Cas9–mediated gene correction (A), AAV8.control.hOTCco donor (equivalent of a gene therapy vector, B), or AAV8.sgRNA1.hOTCco donor for CRISPR-Cas9–mediated gene targeting (C). Liver samples were harvested at 1, 3, 7, 21, and 56 days after vector injection for immunostaining with an OTC antibody. Representative pictures at each time point are shown. Scale bar, 200 μm.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/6/7/eaax5701/DC1

    Fig. S1. Schematic diagrams of the OTC locus and the targeted OTC locus by HDR or by NHEJ are shown.

    Fig. S2. Gene targeting efficiency analysis by ligation-mediated PCR coupled with unique molecular indices (LMU-PCR).

    Table S1. PCR primer sequences for on-target indel analysis and LMU-PCR.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Schematic diagrams of the OTC locus and the targeted OTC locus by HDR or by NHEJ are shown.
    • Fig. S2. Gene targeting efficiency analysis by ligation-mediated PCR coupled with unique molecular indices (LMU-PCR).
    • Table S1. PCR primer sequences for on-target indel analysis and LMU-PCR.

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