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

Abstract

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 proliferate. 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.

Figure 1. In vivo gene correction of the OTC locus in the spf^ash mouse liver by AAV.CRISPR-SaCas9

(a) Schematic diagram of the mouse OTC locus showing the spf^ash mutation and three SaCas9 targets. spf^ash has a G-to-A 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) Flowchart showing the key steps of AAV8.CRISPR-SaCas9-mediated gene correction in the neonatal OTC spf^ash model.

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

AAV8.SaCas9 (5×10¹⁰ GC/pup) and AAV8.sgRNA1.donor (5×10¹¹ GC/pup) were administrated to postnatal day 2 (p2) spf^ash pups via the temporal vein. spf^ash mice were sacrificed at 3 (n=5) or 8 weeks (n=8) after treatment. Untargeted spf^ash mice received AAV8.SaCas9 (5×10¹⁰ GC/pup) and AAV8.control.donor (5×10¹¹ GC/pup) at p2, and livers were harvested 8 weeks post treatment (n=6). Untreated WT (n=3) and spf^ash mice (n=3) were included as controls. (a) Immunofluorescence staining with antibodies against OTC on liver sections from spf^ash 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, spf^ash heterozygous, and spf^ash hemizygous mice. Scale bar, 100μm. (b) Quantification of gene correction based on the percentage of area on liver sections expressing OTC by immunostaining as presented in panel 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 bar, 50μm. (e) OTC enzyme activity in the liver lysate of spf^ash 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 ± SEM are shown. * P < 0.05, ** P < 0.01, *** P < 0.001, Dunnett’s test.

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 spf^ash 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 GC/pup) and AAV8.sgRNA1.donor (5×10¹¹ GC/pup) were administrated to p2 spf^ash pups via 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 ± SEM are shown. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, Dunnett’s test. (c) Quantification of SaCas9 vector genome in liver by qPCR. (d, e) Plasma ammonia levels and survival curves in control or dual AAV vector-treated spf^ash mice after a one-week course of high-protein diet. Seven weeks following neonatal treatment with the dual AAV vectors, mice were given high-protein diet for 7 days. (d) Plasma ammonia levels were measured 7 days after the high-protein diet. Plasma ammonia levels in WT mice (n=13) and AAV8.SaCas9 + AAV8.sgRNA1.donor-treated spf^ash mice (n=13) were significantly lower than untreated spf^ash mice (n=16) after a 7-day high-protein diet. Red squares indicate samples obtained from moribund untreated spf^ash mice 6 days after high-protein diet; red triangle indicates sample obtained from a moribund spf^ash mouse treated with untargeted vector (AAV8.control.donor with no sgRNA1, n=10) 5 days after high-protein diet. ** P< 0.01, **** P< 0.0001, Dunnett’s test. (e) Untreated spf^ash mice (n=20) or spf^ash mice treated with untargeted vectors (AAV8.control.donor, n=13) started to die 3 days 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.

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

Adult spf^ash mice (8–10 weeks old) received an intravenous injection of AAV8.SaCas9 (1×10¹¹ GC) and AAV8.sgRNA1.donor (1×10¹² GC), or higher dose of AAV8.SaCas9 (1×10¹² GC) and AAV8.sgRNA1.donor (5×10¹² GC), 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, 100μm. (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, 50μm. (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 days post vector injection. (e) Change of urine orotic acid levels in adult spf^ash mice after treatment with high-dose gene targeting vectors (n=3 for untreated spf^ash and low-dose groups; n=2 for high-dose groups) (f) Elevation of plasma NH₃ levels in adult spf^ash mice after treatment with high-dose gene targeting vectors (n=3 for each group). Mean ± SEM are shown. *** P < 0.001, **** P < 0.0001, Dunnett’s test.