A dual AAV system enables the Cas9-mediated correction of a metabolic liver disease in newborn mice

Journal name:
Nature Biotechnology
Volume:
34,
Pages:
334–338
Year published:
DOI:
doi:10.1038/nbt.3469
Received
Accepted
Published online

Many genetic liver diseases in newborns cause repeated, often lethal, metabolic crises. Gene therapy using nonintegrating viruses such as adeno-associated virus (AAV) is not optimal in this setting because the nonintegrating genome is lost as developing hepatocytes proliferate1, 2. We reasoned that newborn liver may be an ideal setting for AAV-mediated gene correction using CRISPR-Cas9. Here we intravenously infuse two AAVs, one expressing Cas9 and the other expressing a guide RNA and the donor DNA, into newborn mice with a partial deficiency in the urea cycle disorder enzyme, ornithine transcarbamylase (OTC). This resulted in reversion of the mutation in 10% (6.7–20.1%) of hepatocytes and increased survival in mice challenged with a high-protein diet, which exacerbates disease. Gene correction in adult OTC-deficient mice was lower and accompanied by larger deletions that ablated residual expression from the endogenous OTC gene, leading to diminished protein tolerance and lethal hyperammonemia on a chow diet.

At a glance

Figures

  1. In vivo gene correction of the OTC locus in the spfash mouse liver by AAV.CRISPR-SaCas9.
    Figure 1: In vivo gene correction of the OTC locus in the spfash mouse liver by AAV.CRISPR-SaCas9.

    (a) Schematic diagram of the mouse OTC locus showing the spfash mutation and three SaCas9 targets. spfash has a Gright arrowA mutation at the donor splice site at the end of exon 4 indicated in red on the top strand. The three selected SaCas9-targeted genomic sites (20 bp each) are in blue and underlined with the PAM sequences marked in green. The black line above exon 4 indicates the 1.8-kb OTC donor template. (b) Dual AAV vector system for liver-directed and SaCas9-mediated gene correction. The AAV8.sgRNA1.donor vector contains a 1.8-kb murine OTC donor template sequence as shown in a with the corresponding PAM sequence mutated and an AgeI site inserted. (c) The key steps of AAV8.CRISPR-SaCas9-mediated gene correction in the neonatal OTC spfash model.

  2. Efficient restoration of OTC expression in the liver of spfash mice treated at neonatal stage by AAV8.CRISPR-SaCas9-mediated gene correction.
    Figure 2: Efficient restoration of OTC expression in the liver of spfash mice treated at neonatal stage by AAV8.CRISPR-SaCas9-mediated gene correction.

    AAV8.SaCas9 (5 × 1010 genome copies/pup) and AAV8.sgRNA1.donor (5 × 1011 genome copies/pup) were administrated to postnatal day 2 (p2) spfash pups via the temporal vein. spfash mice were euthanized at 3 (3w; n = 5) or 8 weeks (8w; n = 8) after treatment. Untargeted spfash mice received AAV8.SaCas9 (5 × 1010 genome copies/pup) and AAV8.control.donor (5 × 1011 genome copies/pup) at p2, and livers were harvested 8 weeks after treatment (n = 6). Untreated WT (n = 3) and spfash mice (n = 3) 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 correction. Stained areas typically represent clusters of corrected hepatocytes. Untreated controls show livers from wild-type, spfash heterozygous and spfash hemizygous mice. Scale bar, 100 μm. (b) Quantification of gene correction based on the percentage of area on liver sections expressing OTC by immunostaining as presented in a. (c) Random distribution of clusters of corrected hepatocytes along the portal-central axis shown by double immunostaining against OTC (red) and glutamine synthetase (GS, green), which is a marker of central veins (p, portal vein; c, central vein). Scale bars, 300 μm (upper panel) and 100 μm (lower panel). (d) Groups of corrected hepatocytes expressing OTC (red) shown by immunofluorescence on sections co-stained with fluorescein-labeled tomato lectin (Lycopersicon esculentum lectin, LEL; green), which outlines individual hepatocytes. Scale bars, 50 μm. (e) OTC enzyme activity in the liver lysate of spfash mice at 3 and 8 weeks following dual vector treatment. (f) Quantification of OTC mRNA levels in the liver by RT-qPCR using primers spanning exons 4–5 to amplify wild-type OTC. Mean ± s.e.m. are shown. *P < 0.05, **P < 0.01, ***P < 0.001, Dunnett's test.

  3. Time course of SaCas9 expression following neonatal vector administration and functional improvement following high-protein diet challenge.
    Figure 3: Time course of SaCas9 expression following neonatal vector administration and functional improvement following high-protein diet challenge.

    (a) Immunostaining with antibodies against FLAG on liver sections from an untreated mouse or treated spfash mice at 1, 3 or 8 weeks following neonatal injection of the dual AAV vectors for CRISPR-SaCas9-mediated gene correction. AAV8.SaCas9 (5 × 1010 genome copies/pup) and AAV8.sgRNA1.donor (5 × 1011 genome copies/pup) were administrated to p2 spfash pups through the temporal vein. Nuclear staining of FLAG-tagged SaCas9 were abundant at 1 week (n = 5) but dramatically reduced at 3 weeks (n = 6) and became scarce at 8 weeks (n = 7) after vector injection. Scale bar, 100 μm. (b) Quantification of SaCas9 mRNA levels in liver by RT-qPCR. Mean ± s.e.m. are shown. *P < 0.05, **P < 0.01, Dunnett's test. (c) Quantification of SaCas9 vector genome in liver by qPCR. ***P < 0.001, ****P < 0.0001, Dunnett's test. (d,e) Plasma ammonia levels and survival curves in control or dual AAV vector–treated spfash mice after a 1-week course of high-protein diet. Seven weeks following neonatal treatment with the dual AAV vectors, mice were given high-protein diet for 7 d. (d) Plasma ammonia levels were measured 7 d after the high-protein diet. Plasma ammonia levels in WT mice (n = 13) and AAV8.SaCas9 + AAV8.sgRNA1.donor-treated spfash mice (n = 13) were significantly lower than untreated spfash mice (n = 16) after a 7-day high-protein diet. Red squares indicate samples obtained from moribund untreated spfash mice 6 d after high-protein diet; red triangle indicates sample obtained from a moribund spfash mouse treated with untargeted vector (AAV8.control.donor with no sgRNA1, n = 10) 5 d after high-protein diet. **P < 0.01, ****P < 0.0001, Dunnett's test. (e) Untreated spfash mice (n = 20) or spfash mice treated with untargeted vectors (AAV8.control.donor, n = 13) started to die 3 d after high-protein diet. All WT (n = 13) and AAV8.SaCas9 + AAV8.sgRNA1.donor-treated mice (n = 13) survived. P < 0.05, Mantel-Cox test.

  4. Gene targeting/correction in the liver of spfash mice treated as adults by AAV8.CRISPR-SaCas9 vectors.
    Figure 4: Gene targeting/correction in the liver of spfash mice treated as adults by AAV8.CRISPR-SaCas9 vectors.

    Adult spfash mice (8 to 10 weeks old) received an intravenous injection of AAV8.SaCas9 (1 × 1011 genome copies) and AAV8.sgRNA1.donor (1 × 1012 genome copies), or higher dose of AAV8.SaCas9 (1 × 1012 genome copies) and AAV8.sgRNA1.donor (5 × 1012 genome copies), or untargeted vectors at the equivalent doses. (a) Immunofluorescence staining with antibodies against OTC on liver sections collected at 3 (low-dose, n = 3) or 2 weeks (high-dose, n = 3) after injection. Stained cells typically showed as single corrected hepatocytes. Scale bar, 100 μm. (b) Isolated corrected hepatocytes expressing OTC (red) shown by immunofluorescence on sections co-stained with fluorescein-labeled tomato lectin (LEL; green), which outlines individual hepatocytes. Scale bar, 50 μm. (c) Survival curve of the low-dose cohorts: sgRNA1 (n = 10) or untargeted vector at the same dose (n = 5). (d) Survival curve of the high-dose cohorts: sgRNA1 (n = 5) or untargeted vector at the same doses (n = 5). The experiment was terminated at 14 d after vector injection. (e) Change of urine orotic acid levels in adult spfash mice after treatment with high-dose gene targeting vectors (n = 3 for untreated spfash and low-dose groups; n = 2 for high-dose groups). (f) Elevation of plasma NH3 levels in adult spfash mice after treatment with high-dose gene targeting vectors (n = 3 for each group). Mean ± s.e.m. are shown. ***P < 0.001, ****P < 0.0001, Dunnett's test.

  5. In vitro validation of OTC sgRNAs and donor template.
    Supplementary Fig. 1: In vitro validation of OTC sgRNAs and donor template.

    (a) In vitro validation of sgRNAs targeted to OTC in the MC57G mouse cell line by transient transfection followed by 4-day puromycin enrichment and SURVEYOR nuclease assays. sgRNA1 showed the highest efficiency in inducing indels in the targeted loci and was therefore chosen for subsequent studies. Arrows denote SURVEYOR nuclease cleaved fragments of the OTC PCR products. Results were replicated in 2 independent experiments. (b) In vitro validation of OTC donor template. MC57G cells were transiently transfected with a plasmid co-expressing OTC sgRNA1, SaCas9, and an AgeI restriction site tagged OTC donor plasmid followed by 4-day puromycin enrichment. RFLP analysis was performed following AgeI digestion to detect HDR in vitro. Co-transfection of the AgeI-tagged OTC donor template with an SaCas9 plasmid without OTC sgRNA1 did not result in detectable HDR. Arrows denote AgeI-sensitive cleavage products resulting from HDR. Results were replicated in 2 independent experiments. Indel and HDR frequency were calculated based on band intensities31.

  6. Vector dose optimization to improve in vivo gene correction.
    Supplementary Fig. 2: Vector dose optimization to improve in vivo gene correction.

    Postnatal day 2 spfash pups received temporal vein injection of 5x1010 GC AAV8.SaCas9 and either 5x1010 (n=5), 1x1011 (n=3), or 5x1011 (n=5) GC of AAV8.sgRNA1.donor vector. Liver samples were collected 3 weeks post vector treatment for analysis. (a) Quantification of gene correction based on the percentage of area on liver sections expressing OTC by immunostaining. (b) Quantification of OTC mRNA levels in the liver by RT-qPCR using primers spanning exons 4–5 to amplify wild-type OTC. Mean ± SEM are shown. ** P<0.01, Dunnett’s test.

  7. Time course of gene expression by Western analysis and HDR analysis by RFLP.
    Supplementary Fig. 3: Time course of gene expression by Western analysis and HDR analysis by RFLP.

    (a) HDR analysis by RFLP. OTC target region was PCR amplified from the liver genomic DNA isolated from untreated spfash mice or spfash mice treated with the dual AAV vectors. Untreated spfash control samples were collected at 8 weeks of age. Samples from the treated spfash mice were collected at 1, 3, and 8 weeks (n=3 animals per time point) following neonatal injection of the dual AAV8 vectors. Targeted animals received AAV8.SaCas9 (5x1010 GC/pup) and AAV8.sgRNA1.donor (5x1011 GC/pup). Untargeted animals received AAV8.SaCas9 (5x1010 GC/pup) and AAV8.control.donor (5x1011 GC/pup). AgeI digestion was performed and estimated HDR percentages are shown. (b) Western blot analysis. Liver lysates were prepared from untreated WT and spfash mice or spfash mice treated with the dual AAV vectors for detection of FLAG-SaCas9 and OTC protein.

  8. Examination of liver toxicity in animals treated with AAV8.CRISPR-SaCas9 dual vectors.
    Supplementary Fig. 4: Examination of liver toxicity in animals treated with AAV8.CRISPR-SaCas9 dual vectors.

    (a) Histological analysis on livers harvested 3 and 8 weeks following the dual vector treatment. Scale bar, 100 µm. (b) Liver transaminase levels in untreated spfash mice (n=9) or 8 weeks following dual vector treatment. Untargeted mice received 5x1010 GC AAV8.SaCas9 and 5x1011 of AAV8.control.donor vectors (n=8), while gene-targeted mice received 5x1010 GC AAV8.SaCas9 and 5x1011 GC of AAV8.sgRNA1.donor (n=7). Mean ± SEM are shown. There were no statistically significant differences between groups, Dunnett’s test.

  9. Examination of liver toxicity in adult animals treated with AAV8.CRISPR-SaCas9 dual vectors.
    Supplementary Fig. 5: Examination of liver toxicity in adult animals treated with AAV8.CRISPR-SaCas9 dual vectors.

    (a) Histological analysis on livers harvested 3 weeks (low-dose) or 2 weeks (high-dose) following dual vector treatment. Scale bar, 100 µm. (b) Liver transaminase levels in untreated spfash mice, or 3 weeks following low-dose dual vector treatment, or 2 weeks following high-dose dual vector treatment (n=3 for each group). Low-dose, untargeted mice received 1x1011 GC AAV8.SaCas9 and 1x1012 GC of AAV8.control.donor vectors, while low-dose, gene-targeted mice received 1x1011 GC AAV8.SaCas9 and 1x1012 GC of AAV8.sgRNA1.donor. High-dose, untargeted mice received 1x1012 GC AAV8.SaCas9 and 5x1012 GC of AAV8.control.donor vectors, while high-dose, gene-targeted mice received 1x1012 GC AAV8.SaCas9 and 5x1012 GC of AAV8.sgRNA1.donor. Mean ± SEM are shown. Adult animals received high-dose, gene-targeted vectors showed a trend of elevated ALT and AST levels, although not statistically different when compared with other groups (Dunnett’s test).

  10. Comparison of SaCas9 vector DNA and mRNA levels in the livers of neonatal treated and adult treated mice.
    Supplementary Fig. 6: Comparison of SaCas9 vector DNA and mRNA levels in the livers of neonatal treated and adult treated mice.

    Neonatal spfash mice received 5x1010 GC AAV8.SaCas9 and 5x1011 GC of AAV8.sgRNA1.donor vectors and were sacrificed at 1 (n=5), 3 (n=6), and 8 weeks (n=7) after injection. Low-dose adult spfash mice received 1x1011 GC AAV8.SaCas9 and 1x1012 GC of AAV8.sgRNA1.donor vectors and were sacrificed at 3 weeks (n=3) after injection. High-dose adult spfash mice received 1x1012 GC AAV8.SaCas9 and 5x1012 GC of AAV8.sgRNA1.donor vectors and were sacrificed at 2 weeks (n=3) after injection. (a) Quantification of SaCas9 vector DNA in the liver by qPCR. (b) Quantification of SaCas9 mRNA in the liver by RT-qPCR. Mean ± SEM are shown.

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Author information

  1. These authors contributed equally to this work.

    • Yang Yang &
    • Lili Wang

Affiliations

  1. Gene Therapy Program, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

    • Yang Yang,
    • Peter Bell,
    • Deirdre McMenamin,
    • Zhenning He,
    • John White,
    • Hongwei Yu &
    • James M Wilson
  2. State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, and Collaborative Innovation Center for Biotherapy, Chengdu, Sichuan, China.

    • Yang Yang
  3. Gene Therapy Program, Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

    • Lili Wang
  4. Center for Genetic Medicine Research, Children's Research Institute, Children's National Health System, Washington, DC, USA.

    • Chenyu Xu,
    • Hiroki Morizono &
    • Mark L Batshaw
  5. Department of Stem Cell and Regenerative Biology, Harvard University, Harvard Stem Cell Institute, Cambridge, Massachusetts, USA.

    • Kiran Musunuru
  6. Division of Cardiovascular Medicine, Brigham and Women's Hospital, Boston, Massachusetts, USA.

    • Kiran Musunuru

Contributions

L.W. and J.M.W. conceived this study. L.W., Y.Y. and J.M.W. designed the experiments. Y.Y., P.B., D.M., Z.H., J.W., H.Y. and C.X. performed the experiments. K.M. conducted the bioinformatics analysis of the deep sequencing data. J.M.W., L.W., Y.Y., P.B., H.M., K.M. and M.L.B. wrote and edited the manuscript.

Competing financial interests

J.M. Wilson is an advisor to REGENXBIO, Dimension Therapeutics and Solid Gene Therapy, and is a founder of, holds equity in, and has a sponsored research agreement with REGENXBIO and Dimension Therapeutics; in addition, he is a consultant to several biopharmaceutical companies and is an inventor on patents licensed to various biopharmaceutical companies.

Corresponding author

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Supplementary information

Supplementary Figures

  1. Supplementary Figure 1: In vitro validation of OTC sgRNAs and donor template. (29 KB)

    (a) In vitro validation of sgRNAs targeted to OTC in the MC57G mouse cell line by transient transfection followed by 4-day puromycin enrichment and SURVEYOR nuclease assays. sgRNA1 showed the highest efficiency in inducing indels in the targeted loci and was therefore chosen for subsequent studies. Arrows denote SURVEYOR nuclease cleaved fragments of the OTC PCR products. Results were replicated in 2 independent experiments. (b) In vitro validation of OTC donor template. MC57G cells were transiently transfected with a plasmid co-expressing OTC sgRNA1, SaCas9, and an AgeI restriction site tagged OTC donor plasmid followed by 4-day puromycin enrichment. RFLP analysis was performed following AgeI digestion to detect HDR in vitro. Co-transfection of the AgeI-tagged OTC donor template with an SaCas9 plasmid without OTC sgRNA1 did not result in detectable HDR. Arrows denote AgeI-sensitive cleavage products resulting from HDR. Results were replicated in 2 independent experiments. Indel and HDR frequency were calculated based on band intensities31.

  2. Supplementary Figure 2: Vector dose optimization to improve in vivo gene correction. (31 KB)

    Postnatal day 2 spfash pups received temporal vein injection of 5x1010 GC AAV8.SaCas9 and either 5x1010 (n=5), 1x1011 (n=3), or 5x1011 (n=5) GC of AAV8.sgRNA1.donor vector. Liver samples were collected 3 weeks post vector treatment for analysis. (a) Quantification of gene correction based on the percentage of area on liver sections expressing OTC by immunostaining. (b) Quantification of OTC mRNA levels in the liver by RT-qPCR using primers spanning exons 4–5 to amplify wild-type OTC. Mean ± SEM are shown. ** P<0.01, Dunnett’s test.

  3. Supplementary Figure 3: Time course of gene expression by Western analysis and HDR analysis by RFLP. (52 KB)

    (a) HDR analysis by RFLP. OTC target region was PCR amplified from the liver genomic DNA isolated from untreated spfash mice or spfash mice treated with the dual AAV vectors. Untreated spfash control samples were collected at 8 weeks of age. Samples from the treated spfash mice were collected at 1, 3, and 8 weeks (n=3 animals per time point) following neonatal injection of the dual AAV8 vectors. Targeted animals received AAV8.SaCas9 (5x1010 GC/pup) and AAV8.sgRNA1.donor (5x1011 GC/pup). Untargeted animals received AAV8.SaCas9 (5x1010 GC/pup) and AAV8.control.donor (5x1011 GC/pup). AgeI digestion was performed and estimated HDR percentages are shown. (b) Western blot analysis. Liver lysates were prepared from untreated WT and spfash mice or spfash mice treated with the dual AAV vectors for detection of FLAG-SaCas9 and OTC protein.

  4. Supplementary Figure 4: Examination of liver toxicity in animals treated with AAV8.CRISPR-SaCas9 dual vectors. (91 KB)

    (a) Histological analysis on livers harvested 3 and 8 weeks following the dual vector treatment. Scale bar, 100 µm. (b) Liver transaminase levels in untreated spfash mice (n=9) or 8 weeks following dual vector treatment. Untargeted mice received 5x1010 GC AAV8.SaCas9 and 5x1011 of AAV8.control.donor vectors (n=8), while gene-targeted mice received 5x1010 GC AAV8.SaCas9 and 5x1011 GC of AAV8.sgRNA1.donor (n=7). Mean ± SEM are shown. There were no statistically significant differences between groups, Dunnett’s test.

  5. Supplementary Figure 5: Examination of liver toxicity in adult animals treated with AAV8.CRISPR-SaCas9 dual vectors. (92 KB)

    (a) Histological analysis on livers harvested 3 weeks (low-dose) or 2 weeks (high-dose) following dual vector treatment. Scale bar, 100 µm. (b) Liver transaminase levels in untreated spfash mice, or 3 weeks following low-dose dual vector treatment, or 2 weeks following high-dose dual vector treatment (n=3 for each group). Low-dose, untargeted mice received 1x1011 GC AAV8.SaCas9 and 1x1012 GC of AAV8.control.donor vectors, while low-dose, gene-targeted mice received 1x1011 GC AAV8.SaCas9 and 1x1012 GC of AAV8.sgRNA1.donor. High-dose, untargeted mice received 1x1012 GC AAV8.SaCas9 and 5x1012 GC of AAV8.control.donor vectors, while high-dose, gene-targeted mice received 1x1012 GC AAV8.SaCas9 and 5x1012 GC of AAV8.sgRNA1.donor. Mean ± SEM are shown. Adult animals received high-dose, gene-targeted vectors showed a trend of elevated ALT and AST levels, although not statistically different when compared with other groups (Dunnett’s test).

  6. Supplementary Figure 6: Comparison of SaCas9 vector DNA and mRNA levels in the livers of neonatal treated and adult treated mice. (42 KB)

    Neonatal spfash mice received 5x1010 GC AAV8.SaCas9 and 5x1011 GC of AAV8.sgRNA1.donor vectors and were sacrificed at 1 (n=5), 3 (n=6), and 8 weeks (n=7) after injection. Low-dose adult spfash mice received 1x1011 GC AAV8.SaCas9 and 1x1012 GC of AAV8.sgRNA1.donor vectors and were sacrificed at 3 weeks (n=3) after injection. High-dose adult spfash mice received 1x1012 GC AAV8.SaCas9 and 5x1012 GC of AAV8.sgRNA1.donor vectors and were sacrificed at 2 weeks (n=3) after injection. (a) Quantification of SaCas9 vector DNA in the liver by qPCR. (b) Quantification of SaCas9 mRNA in the liver by RT-qPCR. Mean ± SEM are shown.

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