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.
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We thank Penn Vector Core for supplying vectors, Penn Bioinformatics Core for assistance on deep sequencing data analysis, Y. Zhu and M. Nayalat for help on immunohistochemistry analysis, and L. Mays for assistance on manuscript preparation. This work was supported by National Institute of Child Health and Human Development P01-HD057247 (J.M.W.) and the Kettering Family Foundation (M.L.B.).
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.
Integrated supplementary information
(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.
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.
(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.
Supplementary Figure 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.
Supplementary Figure 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).
Supplementary Figure 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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Yang, Y., Wang, L., Bell, P. et al. A dual AAV system enables the Cas9-mediated correction of a metabolic liver disease in newborn mice. Nat Biotechnol 34, 334–338 (2016). https://doi.org/10.1038/nbt.3469
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